Analyte detection

ABSTRACT

The present disclosure provides methods and/or kits for detecting an analyte in a sample. Some embodiments provide a method for detecting a non-nucleic acid analyte in a sample using a solid substrate comprising a bound immobilization agent and an antibody capture agent and a detectable agent, which can bind to the analyte. The antibody capture agent comprises, at a plurality of sites, a ligand for the immobilization agent. A complex between the analyte, the antibody capture agent and a detectable agent is formed and immobilized on the solid substrate by binding between the immobilization agent and the ligand. In some embodiments, the ligand and the immobilization agent are a binding pair comprising a peptide tag and an anti-peptide tag antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT/AU2010/001517 entitled“Analyte Detection” filed Nov. 12, 2010 and the entire content of thisPCT application is hereby incorporated by reference. This applicationalso claims priority from, and hereby incorporates by reference in theirentirety, each of the following applications: U.S. provisional patentapplication 61/470,359 filed 31 Mar. 2011 entitled “Detection ofAnalytes” and U.S. provisional patent application 61/470,395 filed 31Mar. 2011 entitled “Improved Immunoassay”.

FIELD

The present invention relates to methods for detecting an analyte in asample.

BACKGROUND

Detection of analytes in samples is important in many industriesincluding, for example, research, immunology, water quality assessment,environmental science and engineering, medicine, etc.

Different methods for detecting analytes in samples may be usedincluding, for example, high pressure liquid chromatography (HPLC), massspectrometry and enzyme-linked immunosorbent assays (ELISA). While HPLCand mass spectrometry may be used to detect analytes on the basis ofcharge and/or size, ELISA may be used to detect an analyte based onantigens on the analyte that are recognisable by capture and detectionagents (e.g. antibodies, aptamers, etc.), making it an important assay,especially in the life sciences. ELISA may be used to detect thepresence, absence or the amount of an analyte in a sample.

While ELISA has become a relatively inexpensive detection method,conventional ELISA takes at least 2 hours to complete and generallyincludes at least 2 separate incubation and washing steps. Accordingly,it would be desirable to provide a method for detecting an analyte in asample that takes less time and inputs to perform compared withconventional ELISA, while maintaining or improving the sensitivity ofdetection.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

SUMMARY

The present disclosure provides methods and/or kits for detecting ananalyte in a sample.

In some embodiments, the present disclosure provides a method fordetecting a non-nucleic acid analyte in a sample, the method comprising:

-   -   providing a solid substrate comprising a bound immobilisation        agent;    -   providing a capture agent which can bind the analyte, wherein        the capture agent comprises, at a plurality of sites, a ligand        for the immobilisation agent;    -   providing a detectable agent which can bind to the analyte;    -   contacting the sample, capture agent and detectable agent to        allow the formation of a complex comprising the analyte, capture        agent and detectable agent;    -   contacting the complex with the solid substrate such that the        immobilisation agent may bind the complex via the ligand; and    -   detecting the presence of immobilised complex on the solid        substrate by detection of the detectable agent.

In some embodiments, the present disclosure provides a kit for detectingan analyte in a sample, the kit comprising:

-   -   an assay platform comprising a plurality of reaction vessels,        one or more of the reaction vessels comprising a bound        immobilisation agent;    -   a capture agent which can bind to an analyte, wherein the        capture agent comprises, at a plurality of sites, a ligand for        the immobilisation agent;    -   a detectable agent which can bind to the analyte, wherein the        detectable agent comprises a detectable tag; and    -   instructions for detecting the analyte.

In certain embodiments, the present disclosure provides a method fordetecting one or more analytes in one or more samples, the methodcomprising:

-   -   providing one or more samples comprising one or more analytes to        be detected;    -   providing a single assay platform comprising at least two        reaction vessels, the at least two reaction vessels comprising a        solid substrate comprising the same bound immobilisation agent;    -   providing one or more capture agents, the one or more capture        agents being able to bind to the one or more analytes to be        detected and comprising, at a plurality sites, a ligand for the        immobilisation agent;    -   providing one or more detectable agents, the one or more        detectable agents being able to bind to the one or more analytes        to be detected;    -   contacting in one or more of the at least two reaction vessels,        the one or more samples, the one or more capture agents and the        one or more detectable agents to allow the formation of one or        more complexes comprising an analyte, a capture agent and a        detectable agent;    -   contacting the one or more complexes with the solid substrate        such that the immobilisation agent may bind the one or more        complexes via the ligand; and    -   detecting the presence of one or more immobilised complexes on        the solid substrate by detection of the one or more detectable        agents.

DETAILED DESCRIPTION

The present invention provides a method for detecting an analyte in asample, the method comprising:

-   -   providing a solid substrate comprising a bound immobilisation        agent;    -   providing a capture agent which can bind the analyte, wherein        the capture agent comprises, at a plurality of sites, a ligand        for the immobilisation agent;    -   providing a detectable agent which can bind to the analyte;    -   contacting the sample, capture agent and detectable agent to        allow the formation of a complex comprising the analyte, capture        agent and detectable agent;    -   contacting the complex with the solid substrate such that the        immobilisation agent may bind the complex via the ligand; and    -   detecting the presence of immobilised complex on the solid        substrate by detection of the detectable agent.

Conventional sandwich ELISA generally involves binding of a captureantibody to a solid substrate prior to exposure of the capture antibodyto an analyte. In conventional ELISAs, the capture antibody is bound oradsorbed to a solid substrate in random orientations. As some of theseorientations may mask part, or all, of the analyte binding domain of thecapture agent, not all the capture agent bound to the solid substratemay be available for analyte binding, thereby reducing the efficiency ofthe capture agent and the assay. Furthermore, in some orientations,although the capture agent may still be able to bind to the analyte,subsequent events in the ELISA, such as binding of the detectable agentto the analyte, may be sterically hindered as a result of theorientation of the capture antibody on the solid substrate, therebyreducing the signal generated and hence the sensitivity and efficiencyof the assay.

In contrast, the method of the present invention promotes the formationof a complex between a capture agent, an analyte and a detectable agentprior to or concurrent with contacting the complex with the solidsubstrate.

Without limiting the present invention to any particular mode of action,formation of the complex before or concurrent with binding of thecapture agent to the solid substrate is thought to prevent or inhibitbinding of the capture agent to the solid substrate in an orientationwhich is not amenable to analyte binding. Thus, substantially all of thecapture agent used may be available for analyte binding. Furthermore,the formation of the complex before or concurrent with binding to thesolid substrate may also prevent or reduce steric hindrance of bindingbetween the detectable agent and the analyte, thus promoting increasedsensitivity of the method.

In the method of the present invention, the amount of capture agent thatbinds to the immobilisation agent in an orientation that masks theanalyte binding domain, may be reduced relative to conventional ELISA,that is, formation of the complex promotes the capture agent binding tothe immobilisation agent in a usable orientation.

As a result of more efficient use of the capture agent (as a result ofthe complex formation described above), the amount of capture agent thatis required to produce a given amount of detectable signal may bereduced relative to conventional ELISA. In addition, more efficient useof the capture agent (as a result of the complex formation describedabove) may also lead to a reduction in the area of solid substraterequired to produce a given level detectable signal relative toconventional ELISA.

In embodiments where a peptide tag is used as a ligand and an antipeptide antibody is used as an immobilisation agent (a peptide/antibodycapture system), such a system may provide one or more additionaladvantages.

For example, it has been determined that a peptide/antibody capturesystem has advantages over a streptavidin/biotin capture system, aspeptide/antibody capture systems may provide one or more of increasedsignal, reduced variability and reduced interference depending on thesample type.

In addition, in embodiments utilising a reduced number of washing stepsof the solid substrate, a peptide/antibody capture system may provide anadvantage, particularly in embodiments where the solid substrate is onlywashed after the complex has been immobilised. Further, in embodimentsutilising a reduced time of the assay, a peptide/antibody capture systemmay also provide an advantage to assist in reducing assay time.

Other advantages of a peptide/antibody capture system are describedherein. For example, specific peptides can be designed and prepared thatare not naturally occurring, at least for the organism in which ananalyte is to be detected. Bioinformatics may be used to selectsequences that are unique.

Different peptides may also be selected for different applications orassays. As such, they are readily expandable if more than one affinitysystem is required. For example, in embodiments relating to thedetection of an analyte in different wells of an assay plate, each wellmay be coated with a specific subset of anti-peptide antibodies whichwould allow the specific immobilisation of particular capture antibodiesfrom a mixture of such antibodies with different peptide tags.

Further, in some embodiments the use of a peptide/antibody capturesystem may provide one or more advantages over other types of capturesystems. For example, the use of a peptide/antibody system in certainembodiments may also provide an advantage over capture systems utilisingpoly-charged ligands (for example His tags) and metal ions (for exampleNi²⁺ ions), as the peptide/antibody system may have greater affinityand/or be less likely to be affected by the presence of other chargesmolecules. Similarly, the use of a peptide/antibody system in certainembodiments may provide an advantage over glutathione/GST systems inthat the peptide/antibody capture system also has greater affinity.

A peptide/antibody capture system may also provide in some embodimentsone or more advantages over the use of anti-species antibodies as animmobilisation agent, since the system is then not restricted to the useof species of antibodies immobilised on the surface. For example, ananti-rabbit immobilised antibody can only be used to bind to rabbitcapture antibodies. In addition, anti-species antibodies may suffer fromreduced specificity to the species of antibody they are designed tobind, which may minimize their utility in assays using samplescontaining endogenous antibodies such as serum and plasma, as these willblock the binding of assay antibodies.

Further, a peptide/antibody capture system may also provide in someembodiments one or more advantages over capture systems utilisingimmobilised protein A and/or protein G capture systems. For example,proteins A and G will bind many antibodies in a solution. Protein A andprotein G may also demonstrate reduced utility in samples containingendogenous antibodies, such as serum or plasma, as these will blockbinding of antibodies. In addition, such a capture system may havedisadvantages in embodiments where both the capture agent and thedetectable agent are antibodies, since Protein A or Protein G will notdiscriminate between the capture and detection antibodies, and will bindboth, therefore eliminating the assay discrimination for analyte.

As the method allows detectable signals to be produced with less captureagent and/or reduced solid substrate surface area relative toconventional ELISA, the method may be particularly suitable formicrofluidic systems, where miniaturisation of structures andminimisation of reagents used is desirable. Accordingly, in someembodiments, the method may be performed in a microfluidic system.

Microfluidics deals with the behaviour, precise control and manipulationof fluids that are geometrically constrained to a small, typically inthe low millimeter, or sub-millimeter scale. The behaviour of fluids atthe microscale can differ from ‘macrofluidic’ behaviour in that factorssuch as surface tension, energy dissipation, and fluidic resistancestart to dominate the system. At small scales (channel diameters ofaround 100 nanometers to several hundred micrometers) fluids exhibitspecific properties. For example, the Reynolds number (which comparesthe effect of momentum of a fluid to the effect of viscosity) can becomevery low. A key consequence of this is that fluids, when side-by-side,do not necessarily mix in the traditional sense; molecular transportbetween them must often be through diffusion. Furthermore, highspecificity of chemical and physical properties (concentration, pH,temperature, shear force, etc.) can also be ensured resulting in moreuniform reaction conditions and higher grade products in single andmulti-step reactions.

In addition, as set out above, in microfluidic systems, the surface areaavailable for surface-based reactions, such as the formation of animmobilised complex on a surface, may be limited, thus making the methodof the present invention particularly suited to microfluidic systems.

In light of the above, a “microfluidic system” as referred to hereinrefers to where the method of the present invention is at leastpartially performed in a reaction vessel comprising one or more chambersor channels in which the narrowest dimension is less than 3 mm, lessthan 2 mm or less than 1 mm. Alternatively, or in addition, amicrofluidic system may also include any reaction which occurs in atotal reaction volume of less than 20 μl, less than 10 μl, less than 5μl or less than 1 μl. Examples of microfluidic systems may include, forexample, microfluidic “lab-on-a-chip” type devices; high densitymicrotitre plates, such as 1536, 3456 or 9600 well microtitre plates;microarrays and the like.

As described above, in some embodiments, the complex comprising theanalyte, capture agent and detectable agent is formed prior to bindingbetween the immobilisation agent and the ligand. In some embodiments,the complex may be formed by sequential or concurrent addition of thecapture agent and detectable agent to the analyte prior to contactingthe complex with the immobilisation agent on the solid substrate.

In some embodiments, the complex comprising the analyte, capture agentand detectable agent is formed concurrent with binding between theimmobilisation agent and the ligand. For example, the complex may beformed by adding the capture agent, detectable agent and the analyte tothe solid substrate. In some embodiments, the complex may be formed inpart (e.g. analyte+capture agent or analyte+detectable agent) before itis contacted with the solid substrate and the capture agent ordetectable agent.

The time taken to perform a method for detecting an analyte in a sampleis an important consideration in industry. In this regard, conventionalELISA can be time-intensive. For example, it is not uncommon in aconventional ELISA for incubation steps to be performed after theaddition of each individual component of the ELISA (e.g. the captureantibody, the analyte and the detection antibody). In some embodiments,the present invention minimises the number of incubation steps, as thecomplex of the capture agent, the analyte and the detectable agent maybe formed prior to or concurrent with binding to the solid substrate.Accordingly, in some embodiments, only a single incubation step may berequired.

As a result of the multiple incubation steps and sequential addition ofcomponents, conventional ELISA generally also requires multiple washsteps to remove unbound components after each incubation step. Forexample, it is not uncommon in a conventional ELISA for washing steps tobe performed after binding of a capture antibody to a solid substrate,after addition of an analyte and after addition of a detection antibody.In some embodiments, the method of the present invention allows thenumber of washing steps to be reduced compared with conventional ELISA.For example, as the capture agent, analyte and detectable agent may beadded to the solid substrate at the same time, intermediate washingsteps may be avoided. The reduced number of washes may allow the methodto be performed in a simpler and more time-efficient manner.Furthermore, in some embodiments, the reduced number of washes allowsthe method to be used for capture agents that may have a low bindingaffinity to the analyte, as the reduced amount of washing may reduce oreliminate dissociation between the capture agent and the analyte.

In some embodiments, reducing the number of incubation steps and/orwashing steps that are required may allow the duration of the complex tosolid substrate binding step to be maximised without increasing thetotal duration of the method. Increasing the duration of the complex tosolid substrate binding step may increase the sensitivity of the method.

Notwithstanding, in some embodiments, it may be desirable to washunbound components from the solid substrate after the complex has boundto the solid substrate. Accordingly, in some embodiments, the solidsubstrate may be washed prior to detection of the detectable agent.Washing the solid substrate prior to detection of the detectable agentallows the removal of unbound detectable agent, which can decrease thelevel of background signal and hence improve the sensitivity of theELISA. Methods for washing steps are known in the art and generallyinvolve repeated addition and removal of buffer. For example, washingsteps may be performed as described in Moore et al. AIDS 3(3): 155-163,1989.

As set out above, the method comprises providing a solid substratecomprising a bound immobilisation agent. In this regard, the solidsubstrate comprising the bound immobilisation agent may be any suitablesubstrate for binding the immobilisation agent and permitting detectionof the detectable agent. The solid substrate may, for example, comprisea surface of a multi-well plate (e.g. a microtitre plate), a multi-wellstrip, a bead, a dip stick, a microfluidic device, etc.

In some embodiments, the use of the bound immobilisation agent on thesolid substrate provides flexibility in the selection of the substratethat may be used. For example, the immobilisation agent may allow aparticular capture agent to bind to a substrate (via the immobilisationagent) to which it would otherwise not bind. Moreover, the use of animmobilisation agent-ligand binding pair allows the method to be modularin that a range of capture agents may be produced that bind to aparticular solid substrate by incorporation of a ligand for theimmobilisation agent on the solid substrate into the capture agents.

In some embodiments, the solid substrate may comprise a substance thatpromotes binding of the immobilisation agent or may be treated topromote binding of the immobilisation agent. In some embodiments, thesolid substrate may comprise a plastic surface including, for example, apolystyrene surface, a polyvinyl chloride surface or a cyclo-olefinsurface. In some embodiments, the solid substrate may be transparent orcoloured depending whether the detection method involves a colorimetric,fluorescence or other read out.

In some embodiments, the solid substrate may comprise a hydrophobicsurface.

In some embodiments, the solid substrate may be treated to increase thebinding affinity of the immobilisation agent to the solid substrate. Forexample, the solid substrate may be irradiated or functionalised toallow covalent bonding between the substrate and the immobilisationagent.

As described above, the solid substrate comprises a bound immobilisationagent which is capable of binding a ligand on the capture agent. As canbe appreciated, a range of different immobilisation agent and ligandbinding pairs may be used. In some embodiments, the immobilisation agentand ligand may be interchangeable (i.e. a first compound may be bound tothe solid substrate or the capture agent and a second compound, which ispart of the same binding pair, may be bound to the other).

In some embodiments, the immobilisation agent and ligand binding pairsmay comprise, for example: biotin and avidin or streptavidin (orderivates thereof); a metal chelate (e.g. copper, nickel, cobalt) andHistidine (e.g. histidine tagged proteins); maleic anhydride and amine(e.g. amine containing proteins); or meleimide and sulfhydryls (e.g.sulfhydryl peptides); a FLAG-tag and an anti-FLAG antibody; and thelike.

In some embodiments, the immobilisation agent comprises avidin,streptavidin or derivatives thereof and the ligand comprises biotin orderivates thereof. Derivatives of avidin or streptavidin are known inthe art and may include forms of avidin or streptavidin that have beenmodified to increase their binding affinity to modified and/orunmodified solid substrates or ligands. For example, streptavidin may bemodified to add one or more amine groups, histidine residues orsulfhydryl groups to the molecule. In some embodiments, the derivativeof streptavidin may comprise neutravidin, captavidin or streptavidinmutants (e.g. H127C or S139C).

In some embodiments, where a FLAG-tag is used as a ligand in the methodof the present invention (see later), the corresponding immobilizationagent may comprise an anti-FLAG antibody. Reference herein to an“antibody” may include, for example, monoclonal antibodies, polyclonalantibodies, multivalent antibodies, chimeric antibodies, multispecificantibodies, and antibody fragments that exhibit the desired bindingspecificity to a FLAG-tag. A range of anti-FLAG antibodies could bereadily obtained or produced by a person skilled in the art. However, byway of example, commercially available anti-FLAG antibodies includeSigma-Aldrich product codes F7425, F3040, F1804, F3165, F4042, F2555 andSAB4200071. Some commercially available antibodies recognize theFLAG-tag only in certain positions on a protein, e.g. exclusivelyN-terminal. However, other available antibodies areposition-insensitive.

In some embodiments, hydrophobic or hydrophilic immobilisation agentsmay be passively bound to the hydrophobic or hydrophilic solidsubstrates, respectively. For example, streptavidin (or derivatesthereof) may be passively bound to a hydrophobic solid substrate. Insome embodiments, the solid substrate may comprise a linker whichfacilitates covalent bonding of the immobilisation agents to the solidsubstrate. For example, the linker may comprise glutathione, maleicanhydride, a metal chelate, or meleimide. The immobilisation agent maythen be bound to the solid substrate via the linker.

In some embodiments, the solid substrate comprising the boundimmobilisation agent may be treated with a blocking agent that bindsnon-specifically to and saturates binding sites to prevent unwantedbinding of ligand or other components (e.g. the analyte, capture agentor detectable agent) to the excess sites on the solid substrate. In someembodiments, a blocking agent may be included during the bindingreactions (e.g. bovine serum albumin (BSA), or the like, may be includedduring the formation of the complex). Examples of blocking agents mayinclude, for example, gelatin, BSA, egg albumin, casein, and non-fatmilk. In some embodiments, the solid substrate and/or the boundimmobilisation agent may be treated with the blocking agent prior to theaddition of the capture agent or concurrent with the addition of thecapture agent.

While a single step ELISA has been previously described in Kumada et al.(Journal of Biotechnology 127: 288-299, 2007), such an assay suffersfrom an inability to adequately block the solid substrate, particularlywhen the capture agent has a low binding affinity to the analyte.Inadequate blocking can result in non-specific binding of proteins tothe capture agent or the solid substrate, which can reduce theefficiency of the assay and/or create false or variable signals. Assayssuch as those described in Kumada therefore require a trade-off betweenspecificity and sensitivity and the assay may not be suitable for manysamples and analytes.

In contrast, the utilisation of an immobilisation agent bound to thesolid substrate according to the present invention may allow foradequate blocking of the solid substrate without compromising thesensitivity of the method. In some embodiments, the method may besuitable for high specificity detection of an analyte using a captureagent with a low binding affinity for the analyte.

The ELISA disclosed in Kumada is also limited in relation to the proteinconcentration that may be present in a sample. For example, as describedin Kumada, the immobilisation yield of the capture agent (GST-PS19 orwild-type GST) to a hydrophobic plate drops off significantly in thepresence of a BSA concentration in excess of 0.001 mg/ml. Indeed, in thepresence of a BSA concentration of 0.1 mg/ml, the immobilisation yieldof the capture agent is only around 20%. Similar effects were observedfor the immobilisation of wild-type GST to hydrophilic plates. The assaydisclosed in Kumada is therefore not suitable for samples with moderateto high levels of proteins (e.g. samples comprising serum or celllysates).

In at least some embodiments, the method of the present invention is notso limited. For example, in some embodiments, the utilisation of theimmobilisation agent allows the method to be performed in the presenceof moderate or high protein concentrations. Moderate or high proteinconcentrations may be introduced during blocking steps (as set outabove) or may be included in the sample itself. For example, the samplemay comprise a serum sample which may have a protein concentration up toapproximately 60-80 mg/ml, a cell lysate sample which may have a proteinconcentration of approximately 1-3 mg/ml, or a sample from a cell-basedassay which may include protein contamination from fetal bovine serum(FBS), or the like, which may be used in cell culture media. Proteincontamination from media may account for 1-5% of the final proteincontamination in a cell lysate, which may translate to approximately0.6-4 mg/ml of protein in addition to the cellular protein.

Accordingly, in some embodiments, the sample may comprise a proteinconcentration of more than 0.01 mg/ml, a protein concentration of morethan 0.1 mg/ml, a protein concentration of more than 1 mg/ml, a proteinconcentration of more than 2 mg/ml, a protein concentration of more than10 mg/ml, or a protein concentration of more than 60 mg/ml.

Furthermore, as the immobilisation agent is attached to the solidsubstrate, and the capture agent comprises a ligand for theimmobilisation agent, the potential influence of substrate-reactiveproteins in the sample may be substantially negated. For example,hydrophobic proteins in the sample are not likely to affect the outcomeof the method even if it is performed on a hydrophobic solid substrateas the immobilisation agent is already bound to the solid substrate andthe ligand on the capture agent is specific for the immobilisationagent.

As described above, the capture agent comprises, at a plurality ofsites, a ligand for the immobilisation agent. In some embodiments, theligand may be a part of the capture agent. For example, the captureagent may comprise histidine residues, amine groups or sulfhydryl groupsthat are able to bind to the immobilisation agent.

In some embodiments, the ligand may be bound to the capture agent. Forexample, in some embodiments, the ligand may be amine reactive,carbohydrate reactive, carboxyl reactive, or sulfhydryl reactive andthus may bind to the capture agent via primary amines (e.g. lysine orthe N-terminus), carbohydrate modifications, carboxyl groups (e.g. onaspartic acid residues, glutamic acid residues and the C-terminus), orsulfhydryl groups. In some embodiments, the ligand may compriseiodinatable and/or photoactivatable groups. For example, in embodimentswhereby the capture agent comprises RNA or DNA, the ligand may comprisearyl azide groups that may be converted to highly reactive aryl nitrenewhen exposed to strong visible light or psoralen groups that react withthymine- and other pyrimidine-containing bases when activated to formcovalent bonds (e.g. the ligand may comprise1-[4-Azidosalicylamido]-6-[biotinamido]-hexane or Psoralen-PEG3-Biotin).In some embodiments the ligand may comprise tetrafluorophenyl azide(TFPA) groups that, once activated by UV light, are able to covalentlybind at sites containing C—H or N—H bonds (e.g. the ligand may compriseTFPA-PEG3-Biotin). Methods for labelling proteins, RNA, DNA and othermolecules with the above ligands are generally known in the art and mayinclude methods described by Wong (Chemistry of Protein Conjugation andCross-Linking, CRC Press LLC, 1991).

In some embodiments, the ligand comprises biotin or a derivative thereofincluding, for example, iminobiotin, D-desthiobiotin, DSB-X-biotin,biotin dimers or arylstannyl-biotin trimer. Biotin and derivativesthereof may be bound to the capture agent by biotinylation.Biotinylation reagents and methods for biotinylation of a targetmolecule are known in the art and include those described by Hermanson(Bioconjugate Techniques, Academic Press, 2008). Biotinylation maycomprise, for example, primary amine biotinylation, sulfhydrylbiotinylation, carboxyl biotinylation, or glycoprotein biotinylation.Advantages of biotin or derivatives thereof for labelling the captureagent are the availability of commercial kits, ease of labelling and theability to label the capture agent at a plurality of sites.

In some embodiments, the ligand for the immobilisation agent comprises aFLAG-tag. FLAG-tag, or FLAG octapeptide, is a polypeptide protein tagthat can be conjugated to a protein (such as an antibody) or added to aprotein using Recombinant DNA technology. A FLAG-tag can be used in manydifferent assays that require recognition by an antibody. Adding aFLAG-tag to a protein allows the protein to be bound and/or immobilisedby an antibody against the FLAG sequence. The peptide sequence of theFLAG-tag is DYKDDDDK (SEQ ID NO: 1). In some embodiments, a FLAG-tag mayalso be used in conjunction with other affinity tags for example apolyhistidine tag (His-tag), HA-tag or myc-tag. The FLAG-tag was thefirst example of a fully functional epitope tag to be published in thescientific literature (see Hopp et al., Bio/Technology 6: 1204-1210,1988). Its structure has been optimized for compatibility with theproteins it is attached to, in that it is more hydrophilic than othercommon epitope tags and therefore less likely to denature or inactivateproteins to which it is appended. In addition, it can be removed readilyfrom proteins by treatment with the specific proteinase, enterokinase(Enteropeptidase).

In addition to comprising a ligand for the immobilisation agent at aplurality of sites, the capture agent must be capable of binding to theanalyte. In some embodiments, the capture agent may comprise anantibody, an aptamer, or a protein receptor or ligand (or bindingfragment thereof). Similarly, in some embodiments, the detectable agentwhich can also bind to the analyte may comprise an antibody, an aptamer,or a protein receptor or ligand (or binding fragment thereof).

Reference herein to an “antibody” may include, for example, monoclonalantibodies, polyclonal antibodies, multivalent antibodies, chimericantibodies, multispecific antibodies, and antibody fragments thatexhibit the desired binding specificity. Antibodies to specific analytesmay be obtained commercially or generated by methods known in the art.For example, antibodies to specific analytes may be prepared usingmethods generally disclosed by Howard and Kaser (Making and UsingAntibodies: a Practical Handbook, CRC Press, 2007).

Aptamers used as the capture agent or detectable agent may be obtainedcommercially or generated by methods known in the art. For example,aptamers to specific analytes may be prepared using methods generallydisclosed by Mascini (Aptamers in Bioanalysis, John Wiley & Sons Inc,2009).

Protein receptors or ligands that may be used as the capture agent ordetectable agent may comprise the whole receptor or ligand or a fragmentthereof (e.g. a fragment comprising a binding domain of the receptor orligand). In some embodiments, the receptor or ligand (or fragmentthereof) may comprise a fusion protein. Fusion partners may include, forexample, fluorescent fusion partners (e.g. GFP), immunoglobulin fusionpartners, etc. Fusion partners and methods for preparing fusion proteinsare known in the art and may include those described by Sambrook andRussell (Molecular Cloning: A Laboratory Manual, Volume 3, Cold SpringHarbor Laboratory Press, 2001). In some embodiments, the fusion partnermay act to stabilise the receptor or ligand (or fragment thereof),provide a detectable signal (e.g. for fluorescent fusion partners) orprovide a target for antibody, or other, binding or immobilisation.

In some embodiments, the detectable agent may comprise a detectable tag.In some embodiments, the detectable tag may be applied to the detectableagent (e.g. bound to the detectable agent) or may be part of thedetectable agent (e.g. the detectable agent may include the detectabletag as a fusion partner, a labelled amino acid or labelled nucleotide).

Examples of suitable detectable tags include antigens, enzymes,fluorophores, quenchers, radioactive isotopes, luminescent labels,nucleic acids capable of PCR amplification and the like. It will beappreciated that the detectable tag may be detected directly orindirectly via a further molecule that can produce a detectable signal.

Antigens that may be used as a detectable tag may include, for example,any antigenic component of the detectable agent that may be targeted bya secondary detectable agent. For example, in some embodiments, asecondary antibody may be used to detect an antigen on a detectableagent. The secondary antibody may, for example, be fluorescently orenzymatically labelled. In embodiments where the detectable agent is aprimary antibody (ie. an analyte binding antibody), the secondaryantibody may have binding affinity to an antigen on the primaryantibody. For example, the antigen may be derived from the host in whichthe detectable agent was raised.

Enzymes that may be used as detectable tags include, for example,enzymes that result in the conversion of a substrate into a detectableproduct (generally resulting in a change in colour or fluorescence orgeneration of an electrochemical signal). Such enzymes may include, forexample, horseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, acetylcholinesterase, luciferase, or catalase.Depending on the enzyme and substrate used, detection may be performedwith a spectrophotometer, fluorometer, luminometer, electrochemicaldetection means.

Radioactive isotopes that may be used as detectable tags include, forexample, ³H, ¹⁴C, ³²P, ³⁵S, or ¹³¹I. The radioisotope may be conjugatedto a detectable agent or incorporated into a detectable agent bytranslation of mRNA encoding the detectable agent in the presence ofradiolabelled amino acids. Radioisotopes and methods for conjugatingradioactive isotopes to molecules such as proteins are known in the artand include methods discussed by Slater (Radioisotopes in Biology: APractical Approach, Oxford University Press, 2002). Radioisotopes may bedetected using gamma, beta or scintillation counters.

Fluorophores that may be used as detectable tags include, for example,resorufin, fluorescein (fluorescein isothiocyanate, FITC), rhodamine(tetramethyl rhodamine isothiocyanate, TRITC), green fluorescent protein(GFP), phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrinand phycoerythrocyanin, or derivatives of any of the foregoing). In someembodiments, the detectable tag may be part of the detectable agent(e.g. in the form of a fusion protein or a protein comprisingfluorescent amino acids). Fluorophores may be subjected to appliedstimulation (e.g. light of a suitable excitation wavelength) to promotefluorescence.

Alternatively, stimulation may be provided by a fluorescence resonanceenergy transfer (FRET) partner (i.e. a donor molecule). When thefluorophore comes into close vicinity to the FRET partner (e.g. duringformation of the complex), the fluorophore may become excited by theFRET partner and fluoresce (i.e. the fluorophore is an acceptormolecule). FRET donors may include luminescent and/or fluorescentagents.

In some embodiments, the detectable tag may comprise a quencher.Quenchers are able to absorb excitation energy from fluorophores and maybe used to suppress the fluorophore's emission when in close proximity.In this regard, the reaction is similar to a FRET reaction, except thatthe readout is a loss of fluorescence.

In embodiments which use a quencher or FRET fluorophore as thedetectable tag, an interacting quencher or fluorophore may be providedon, or integrated with, the solid substrate and/or capture agent. Forexample, in embodiments where the detectable tag is a fluorophore, thesubstrate or capture agent may include a FRET partner (either donor oracceptor) or a quencher. Conversely, in embodiments where the detectabletag is a quencher, the solid substrate or capture agent may include asuitable fluorophore. Detection of an analyte in a sample may then bedetermined by a loss or gain in fluorescence via interaction between thefluorophore and quencher or between the FRET donor and acceptor.

In some embodiments, detecting the presence of the immobilised complexon the solid substrate may utilise time-resolved fluorescence (TRF) andFRET technologies. For example detecting the presence of the immobilisedcomplex on the solid substrate may involve a TRF-FRET technology asdescribed in EP 569,496, U.S. Pat. No. 5,527,684 or U.S. Pat. No.6,861,264.

In some embodiments, the detectable tag may comprise a bead thatcomprises a quencher, fluorophore, or FRET donor or acceptor. In someembodiments, the solid substrate and detectable agent may compriselabelled beads which interact via FRET.

In some embodiments, the solid substrate and detectable agent maycomprise labelled beads as part of a chemical transfer proximity basedassay such as the SureFire® detection assay described by Osmond et al.(J. Biomol. Screen. 10(7): 730-737, 2005).

Luminescent compounds that may be used as detectable tags include, forexample, chemiluminescent and bioluminescent compounds. These compoundsmay be used to label the detectable agent. The presence of thechemiluminescent-tag may be determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of useful chemiluminescent labelling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. The presence of a bioluminescentantibody is determined by detecting the presence of luminescence.Examples of bioluminescent compounds include luciferin, luciferase andaequorin.

Nucleic acids that may be used as detectable tags include any suitablenucleic acid that is capable of PCR amplification and/or hybridisationto a probe. Preferably the nucleic acid is of a length sufficient toallow binding of a forward and/or a reverse primer. The nucleic acid tagmay be included in an aptamer or bound to a protein. Bound complex maybe detected by performing a PCR reaction, whereby the nucleic acid tagis amplified and measured, or using a labelled nucleic acid probe with acomplementary sequence to at least a portion of the nucleic acid tag.Methods of preparing and binding detectable nucleic acid tags to captureagents or detectable agents (e.g. proteins) are known in the art andinclude methods described in US 2009/0053701.

As set out above, the present invention provides a method for detectingan analyte in a sample.

In some embodiments, the method for detecting an analyte in a sample maycomprise a qualitative determination of whether the analyte is presentor absent in the sample. In some embodiments, the method may comprise aquantitative assessment of the levels of the analyte in the sample.

The sample may be any mixture, composition, solution, etc. that may ormay not contain an analyte. In some embodiments, the sample may comprisea laboratory sample, medical sample, water sample, food sample,agricultural sample, etc. As described above, in some embodiments, thesample may comprise a serum sample, a serum containing sample or a celllysate.

In some embodiments, the sample may be pretreated before being used inthe method (i.e. the sample may be precleared, concentrated, diluted, orprocessed to remove one or more components or impurities from the sampleusing methods known in the art).

The analyte may be any analyte in a sample against which an antibody,aptamer or other capture agent and detectable agent are able to bind.For example, the analyte may comprise a microbe, a virus, a protein, anucleic acid, a macromolecule, a small molecule, a drug, etc. In someembodiments, the analyte comprises a protein.

In some embodiments, the analyte may comprise a particular form or stateof a protein or other molecule. For example, in some embodiments, themethod may be used to detect a protein that is phosphorylated,methylated, glycosylated, etc. In these embodiments, at least one of thecapture agent and the detectable agent should have specificity to onlyone form of the protein (e.g. the capture agent may only bind to thephosphorylated form of the protein and not to the unphosphorylated formof the protein).

In some embodiments, the analyte may comprise a phosphoprotein. A rangeof phosphoproteins are known including, for example, phosphorylated ERK,S6 p240/44, AKT pT308 or AKT pS473. In some embodiments, the bindingsite of at least one of the capture agent and the detectable agentcomprises a phosphorylation site binding domain. At least one of thecapture agent and the detectable agent may be specific for thephosphorylated or unphosphorylated form of the protein.

Some embodiments of the present disclosure are directed to methodsand/or kits for detecting an analyte in a sample that have one or morecombinations of advantages. For example, some of the advantages of themethods and/or kits disclosed herein include: reducing the time taken todetect the analyte; reducing the number of incubation steps; reducingthe number of step and/or duration for washing of the solid substrate;providing reliable performance; eliminating the need for pre-incubation;reducing the number of dispensing steps; reducing the number ofaspiration steps; providing a simple easy to use assay; being suitablefor microfluidic systems and/or other automated systems; reducing costsin materials; reducing costs in time needed to perform the assay;reducing handling costs; reducing handling errors; reducing the cost ofthe materials needed; measuring different analytes on one plate; beingcompatible for use with most standard plate readers; providing improvedsensitivity; ability to tolerate samples with moderate to high levels ofproteins; allowing the use of antibodies that have a low bindingaffinity to an analyte; improving the ability to detect analytes in avariety of biological milieu; allowing the use of lower concentration ofantibodies to an analyte; and providing kits and/or assay platforms(such as microtitre plates) that are easy to manufacture and/or lesscostly to manufacturer.

For example, the methods and/or kits of the present disclosure mayresult in improvement in the number of handling and/or washing stepsrequired during an assay. In addition, the methods and/or kits of thepresent disclosure may in some embodiments result in an improvement inthe time required to reliably detect the analyte.

Further, the improvements in handling and washing provide a number ofadvantages over previous assays, for example assays for detecting ananalyte utilising an antibody (or an antigen binding part thereof) tocapture an antigen. Such improvements result in an assay that has alower cost to perform and which provides more consistent results overprevious assays. For example, in embodiments utilising apeptide/antibody capture system or a steptavidin/biotin capture system,such systems may assist in reducing the washing of the solid substrate.

By way of illustration, ELISA can take up to 6 hours to complete andconsist of at least 2 separate incubation and washing steps. Otherenzyme-linked immunosorbent assays may generally take over 2 hours tocomplete and also requires at least 2 separate incubation and washingsteps. The methods and/or kits of the present disclosure allow the assayto be performed in a shorter time and in some embodiments, allows asingle incubation, one wash assay to be performed that is superior toprevious ELISAs. For example, the reduction in handling steps allows areduction in common sources of variation that are introduced by multiplehandling steps, plate washing, and extra pipetting steps. Therefore,such previous assays require more handling, take more time and/or usemore product resources and can result in greater costs.

In addition, as a result of the multiple incubation steps and sequentialaddition of components ELISA generally also require multiple wash stepsto remove unbound components after each incubation step. For example, itis not uncommon in an ELISA for washing steps to be performed afterbinding of a capture antibody to a solid substrate, after addition of ananalyte, and after addition of a detection antibody.

In some embodiments, the methods and/or kits of the present disclosureallow the number of washing steps to be reduced compared with previousassays. For example, in some embodiments the capture agent, the analyteand the detectable agent may be added to the solid substrate at the sametime, or substantially the same time, intermediate washing steps may beavoided. The reduced number of washes may allow the methods to beperformed in a simpler and more time-efficient manner. Furthermore, insome embodiments the reduced number of washes allows the methods and/orkits to be used for capture agents that may have a low binding affinityto the analyte, as the reduced amount of washing may reduce and/oreliminate dissociation between the capture agent and the analyte.

As described herein, the methods and/or kits of the present disclosureresult in a number of advantages over traditional assays.

Some embodiments of the methods and/or kits of the present disclosureallow the time to detect an analyte to be reduced.

Some embodiments provide methods and/or kits for detecting an analyte,wherein the detection of the analyte is achieved in a time of less than120 minutes. Some embodiments provide methods and/or kits for detectingan analyte, wherein the detection of the analyte is achieved in a timeof less than 30, 40, 50, 60, 70, 80 or 90 minutes. In some embodiments,the detection of the analyte is achieved in a time range of 30 to 90minutes, 30 to 80 minutes, 30 to 70 minutes, 30 to 60 minutes, 40 to 80minutes, 40 to 70 minutes, 40 to 60 minutes, 50 to 80 minutes, 50 to 70minutes, or 50 to 60 minutes. In some embodiments, the detection of theanalyte is achieved in a time of at least 30, 40, 50, 60, 70, 80 or 90minutes.

In some embodiments, the detection of the analyte is achieved in a timeof less than 30 minutes. In some embodiments, the detection of theanalyte is achieved in a time of less than 25, 15, 10 or 5 minutes. Insome embodiments, the detection of the analyte is achieved in a timerange of 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 5 to 15minutes, 10 to 30 minutes, 10 to 25 minutes, 10 to 15 minutes, 15 to 30minutes, 15 to 25 minutes, 15 to 20 minutes, 20 to 30 minutes, or 20 to25 minutes. In some embodiments, the detection of the analyte isachieved in a time of at least 5, 10, 15, 20, 25, or 30 minutes.

Some embodiments provide methods and/or kits for detecting an analyte,wherein the detection of the analyte is achieved in a time of less than120 minutes from contacting the complex with the solid substrate.

Some embodiments provide methods and/or kits for detecting an analyte ina sample, wherein the detection of the analyte is achieved in a time ofless than 30, 40, 50, 60, 70, 80 or 90 minutes from contacting thecomplex with the solid substrate. In some embodiments, the detection ofthe analyte is achieved in a time range of 30 to 90 minutes, 30 to 80minutes, 30 to 70 minutes, 30 to 60 minutes, 40 to 80 minutes, 40 to 70minutes, 40 to 60 minutes, 50 to 80 minutes, 50 to 70 minutes, or 50 to60 minutes, from contacting the complex with the solid substrate. Insome embodiments, the detection of the analyte is achieved in a time ofat least 30, 40, 50, 60, 70, 80 or 90 minutes from contacting thecomplex with the solid substrate.

In some embodiments, the detection of the analyte is achieved in a timeof less than 30 minutes from contacting the complex with the solidsubstrate. In some embodiments, the detection of the analyte is achievedin a time of less than 25, 15, 10 or 5 minutes contacting the complexwith the solid substrate. In some embodiments, the detection of theanalyte is achieved in a time range of 5 to 30 minutes, 5 to 25 minutes,5 to 20 minutes, 5 to 15 minutes, 10 to 30 minutes, 10 to 25 minutes, 10to 15 minutes, 15 to 30 minutes, 15 to 25 minutes, 15 to 20 minutes, 20to 30 minutes, or 20 to 25 minutes from contacting the complex with thesolid substrate. In some embodiments, the detection of the analyte isachieved in a time of at least 5, 10, 15, 20, 25, or 30 minutes fromcontacting the complex with the solid substrate.

Some embodiments provide methods and/or kits for detecting an analyte,wherein the detection of the analyte is achieved in a time of less than120 minutes from contacting the sample, the capture agent, thedetectable agent and the solid substrate.

Some embodiments provide methods and/or kits for detecting an analyte ina sample, wherein the detection of the analyte is achieved in a time ofless than 30, 40, 50, 60, 70, 80 or 90 minutes from contacting thesample, the capture agent, the detectable agent and the solid substrate.In some embodiments, the detection of the analyte is achieved in a timerange of 30 to 90 minutes, 30 to 80 minutes, 30 to 70 minutes, 30 to 60minutes, 40 to 80 minutes, 40 to 70 minutes, 40 to 60 minutes, 50 to 80minutes, 50 to 70 minutes, or 50 to 60 minutes, from contacting thesample, the capture agent, the detectable agent and the solid substrate.In some embodiments, the detection of the analyte is achieved in a timeof at least 30, 40, 50, 60, 70, 80 or 90 minutes from contacting thesample, the capture agent, the detectable agent and the solid substrate.

In some embodiments, the detection of the analyte is achieved in a timeof less than 30 minutes from contacting the sample, the capture agent,the detectable agent and the solid substrate. In some embodiments, thedetection of the analyte is achieved in a time of less than 25, 15, 10or 5 minutes from contacting the sample, the capture agent, thedetectable agent and the solid substrate. In some embodiments, thedetection of the analyte is achieved in a time range of 5 to 30 minutes,5 to 25 minutes, 5 to 20 minutes, 5 to 15 minutes, 10 to 30 minutes, 10to 25 minutes, 10 to 15 minutes, 15 to 30 minutes, 15 to 25 minutes, 15to 20 minutes, 20 to 30 minutes, or 20 to 25 minutes from contacting thesample, the capture agent, the detectable agent and the solid substrate.In some embodiments, the detection of the analyte is achieved in a timeof at least 5, 10, 15, 20, 25, or 30 minutes from contacting the sample,the capture agent, the detectable agent and the solid substrate.

As described, in some embodiments the reduced number of washes allowsthe methods and/or kits to be used for antibodies (capture antibodiesand/or detectable antibodies) that may have a low or lower, bindingaffinity to the analyte, as the reduced amount of washing may reduceand/or substantially eliminate dissociation between the antibody and theanalyte. In some embodiments, the methods and/or kits may be used with acapture agent having a Kd for binding with the analyte of greater than10⁻⁶M. In further embodiments, the capture agent has a Kd for bindingwith the analyte of greater than 10⁻⁷M, 10⁻⁸M or 10⁻⁹M. In certainembodiments, the Kd is in the range from 10⁻⁸M to 10⁻¹²M.

Further, in previous assays that involve binding of a capture antibodyto a solid substrate prior to exposure of the capture antibody to ananalyte, the capture antibody is bound or adsorbed to a solid substratein random orientations. As some of these orientations may mask part, orall, of the analyte binding domain of the capture agent, some of thecapture agent bound to the solid substrate may not be available foranalyte binding, thereby reducing the efficiency of the capture agentand the assay. Furthermore, in some orientations, although the captureagent may still be able to bind to the analyte, subsequent events suchas binding of the detectable agent to the analyte, may be stericallyhindered as a result of the orientation of the capture antibody on thesolid substrate, thereby reducing the signal generated and hence thesensitivity and efficiency of the assay.

In contrast, in some embodiments the method of the present disclosuremay promote the formation of a complex between a capture agent, ananalyte and a detectable agent before or concurrent with contacting thecomplex with the solid substrate, which may prevent or inhibit bindingof the capture agent to the solid substrate in an orientation which isnot amenable to analyte binding. Thus, a greater proportion of thecapture agent used may be available for analyte binding. Further, alower concentration of capture agent (e.g. antibody) can be utilisedwithout comprising sensitivity and/or specificity.

As described herein, some embodiments are directed to methods and/orkits for detecting an analyte in a sample with improved sensitivityand/or specificity. Some embodiments are directed to methods and/or kitswherein sensitivity is improved by the formation of a complex comprisingthe analyte, the capture agent and the detectable agent, before orconcurrent with contacting the complex with the solid substrate.

As described herein, the methods and/or kits of the present disclosureallow for the detection of an analyte in a sample. In this regard, itwill be understood that some embodiments of the present disclosurerepresent an immunoassay. Some embodiments of the present disclosurerepresent a sandwich assay, in which the analyte is bound to a captureantibody and to an antibody detectable agent.

As described herein, in some embodiments the methods and/or kits maycomprise a qualitative determination of whether the analyte is presentor absent in the sample.

As described herein, in some embodiments the methods and/or kits maycomprise a quantitative assessment of the levels of the analyte in thesample. For example, in some embodiments the methods and/or kits allowfor the quantification of the concentration of the analyte in thesample. Methods for the calculation of the concentration of an analyteare known.

In some embodiments, the method for detecting an analyte comprises animmunoassay. In some embodiments, the immunoassay comprises anon-competitive immunoassay. In some embodiments, the immunoassaycomprises a competitive immunoassay. In some embodiments, theimmunoassay comprises a combination of both a non-competitive and acompetitive immunoassay.

In some embodiments, the analyte comprises one or more antigenic sitesthat allow the analyte to be bound by an antibody capture agent and/oran antibody detectable agent.

In some embodiments, the analyte comprises a non-nucleic acid analyte.

Some embodiments are based on the capture of the analyte by the captureagent via a mechanism that is not substantially based on nucleicacid-nucleic acid interactions, such as a binding based on complementarybase pairing. That being said, it will be understood that in someembodiments, the analyte may comprise a nucleic acid component. In someembodiments the binding of the analyte by the capture agent issubstantially based on hydrophobic, hydrophilic,polyanionic-polycationic, van der Waals, or combinations thereof,substantially exclusive of nucleic acid-nucleic acid interactions.

As described herein, examples of analytes that may be detected by themethods of the present disclosure comprise a microbe, a virus, aprotein, a macromolecule, a small molecule, a drug or combinationsthereof. Other types of analyte are contemplated.

In some embodiments, the analyte may comprise a component of a cellsignalling pathway, a cytokine, a tumour suppressor, an antibody or afragment thereof, or combinations thereof.

As described herein, in some embodiments the analyte may comprise aparticular form or state of a molecule, such as a protein. For example,in some embodiments, the method may be used to detect a protein that isphosphorylated, methylated, glycosylated or combinations thereof. Inthese embodiments, at least one of the capture agent and the detectableagent may have specificity to only one form of the protein (for examplethe capture agent may only bind to the phosphorylated form of theprotein and not to the unphosphorylated form of the protein).

As described herein, in some embodiments, the analyte may comprise aphosphoprotein. Examples of phosphoproteins comprise phosphorylated ERK,S6 p240/44, AKT pT308 or AKT pS473.

In some embodiments, the analyte is selected from the group consistingof phospho-ERK 1/2; total ERK 1/2; phospho-Akt 1/2/3; total Akt 1/2/3;phospho-NF-Kβ p65; total NF-Kβ p65; phospho-1-kβα; total-kβα;phospho-STAT3; total STAT3; phospho-STAT5 A/B; phospho-JNK 1/2/3; totalJNK 1/2/3; phospho-p38 MAPKα; total p38 MAPKα; phospho-p53; total p53;phospho-p70S6K; total p70S6K; and GAPDH.

In some embodiments, the analyte is present in the sample at aconcentration of 100 ng/ml or less, 10 ng/ml or less, 1 ng/ml or less,100 pg/ml or less, or 10 pg/ml or less, 1 pg·ml or less, 100 fg/ml orless, 10 fg/ml or less, or 1 fg/ml or less. In some embodiments, theanalyte is present in the sample at a concentration of greater than 100ng/ml, greater than 10 ng/ml, greater than 1 ng/ml, greater than 100pg/ml or, greater than 10 pg/ml, greater than 1 pg/ml, greater than 100fg/ml, greater than 10 fg/ml or greater than 1 fg/ml. In someembodiments the analyte is present in the sample at a concentration ofbetween 1 fg/ml to 100 ng/ml, 1 fg/ml to 10 ng/ml, 1 fg/ml to 1 ng/ml,10 fg/ml to 100 ng/ml, 10 fg/ml to 10 ng/ml, 10 fg/ml to 1 ng/ml, 100fg/ml to 100 ng/ml, 100 fg/ml to 10 ng/ml, 100 fg/ml to 1 ng/ml, 1 pg/mlto 100 ng/ml, 1 pg/ml to 10 ng/ml, or 1 pg/ml to 1 ng/ml.

As described herein, the present disclosure provides methods and/or kitsfor detecting an analyte in a sample. For example, the sample may be amixture, composition, solution, that may or may not contain an analyte.

In some embodiments, the sample comprises one or more samples. In someembodiments, the sample comprises one or more samples comprising one ormore analytes to be detected.

In some embodiments, the sample comprises a laboratory or researchsample, a medical sample, a biological sample, a cell sample, a watersample, a food sample, an agricultural sample, and/or a derivative ofthese samples or combinations thereof.

In some embodiments, the sample comprises a medical sample or a cellsample, such as a blood sample, a serum sample, a urine sample, a milksample, a cell lysate, a derivative of these samples and/or combinationsthereof.

In some embodiments, the sample may be pre-treated before being used.For example, the sample may be pre-cleared, concentrated, diluted,induced, pre-treated or processed to remove one or more components orimpurities from the sample using known methods.

In some embodiments, the sample may comprise a protein concentration ofmore than 0.01 mg/ml, a protein concentration of more than 0.1 mg/ml, aprotein concentration of more than 1 mg/ml, a protein concentration ofmore than 2 mg/ml, a protein concentration of more than 10 mg/ml, or aprotein concentration of more than 60 mg/ml. In some embodiments, thesample may comprise a protein concentration of less than 0.01 mg/ml, aprotein concentration of less than 0.1 mg/ml, a protein concentration ofless than 1 mg/ml, a protein concentration of less than 2 mg/ml, aprotein concentration of less than 10 mg/ml, or a protein concentrationof less than 60 mg/ml. These protein concentrations demonstrate that insome embodiments the methods and/or kits of the present disclosure arecompatible for analyte detection in a range of biological milieu, suchas cellular lysates, and/or serum.

In some embodiments, the methods and/or kits comprise one or moresamples comprising one or more analytes to be detected. In someembodiments, a sample may comprise one or more analytes to be detected.

In some embodiments, the methods and/or kits comprises providing areaction vessel.

Examples of reaction vessels include a test tube, a micro centrifugetube, a well, or a flask. In some embodiments, the reaction vesselcomprises a well of a multi-well plate, such as a microtitre plate, or awell or surface of a microfluidic device.

In some embodiments, the methods and/or kits comprise an assay platform.In some embodiments, the assay platform comprises one or more reactionvessels.

In some embodiments, the one or more reaction vessels comprise one ormore solid substrates. The one or more solid substrates may comprise oneor more bound immobilisation agents. In some embodiments, one or morereaction vessels comprise one or more bound immobilisation agents. Insome embodiments, the assay platform comprises one or more reactionvessels comprising one or more solid substrates. In some embodiments,the assay platform comprises a multi-well plate, such as a microtitreplate. In some embodiments the one or more reaction vessels comprise oneor more wells of a multi-well plate, such as a microtitre plate. In someembodiments, the assay platform comprises a plurality of reactionvessels comprising a solid substrate comprising the bound immobilisationagent. It will be appreciated that for an assay platform comprising aplurality of reaction vessels, some of the plurality of reaction vesselsmay comprise one or more reaction vessels comprising a boundimmobilisation agent and one or more reaction vessels that do notcomprise a bound immobilisation agent.

In some embodiments, the methods and/or kits comprise providing a singlereaction vessel. In some embodiments, the use of a single reactionvessel for performing the steps in the method onward from the contactingof the sample, the antibody capture agent, the detectable agent and thesolid substrate may reduce the handling steps involved in the method ascompared to previous assays and thereby provide an improvement over suchassays, including the ability to provide more consistent results oversuch assays.

In some embodiments, more than one analyte may be detected in onereaction vessel. In some embodiments, one analyte is detected in asample. In some embodiments, one or more analytes are detected in asample. In some embodiments, at least two analytes are detected in asample.

In some embodiments the detection of more than one analyte may beachieved by providing several target-specific antibody capture agents tothe reaction vessel, in combination with providing their respectivedetectable agents. For example, in some embodiments where eachdetectable agent is an antibody, each antibody may be conjugated to adifferent detectable tag, such as an enzyme, a fluorophore, alanthanide, a chelate or combinations thereof.

In some embodiments, the methods and/or kits of the present disclosureprovide one or more capture agents, the one or more capture agents beingable to bind to one or more analytes to be detected and comprising, at aplurality sites, a ligand for the immobilisation agent.

In some embodiments, the use of a biotin-steptavidin/avidin capturesystem in conjunction with no additional washing of the solid substrateafter contacting of the solid substrate with any one or more of thesample, the capture agent and the detectable agent may provide anadvantage to the detection of more than one analyte.

As described herein, in some embodiments a reaction vessel comprises thesolid substrate. In such embodiments, the solid substrate may be part ofthe reaction vessel. For example, the solid substrate may be integralwith substantially all or part of the reaction vessel, and/or the solidsubstrate may form part of the surface of the reaction vessel (such asthe surface of a well of a microtitre plate) or may be attached to thereaction vessel. Other combinations are also contemplated.

In some embodiments, the solid substrate is separate to the reactionvessel. In these embodiments, the solid substrate may be mobilisable andmay be added to the reaction vessel.

For example, the solid substrate may be a bead, an affinity matrix, aresin, a gel, a slurry, a strip, or a dip stick. Combinations ofdifferent types of solid substrates are also contemplated.

In some embodiments, the bead is a magnetic bead. Methods for the use ofmagnetic beads are known in the art.

In some embodiments, the immobilisation agent is bound to the solidsubstrate by a covalent attachment to the solid substrate. For example,in some embodiments wherein the solid substrate is a bead, theimmobilisation agent may be bound to the bead by a covalent attachmentto the bead.

In some embodiments, the immobilisation agent is bound to the solidsubstrate via a non-covalent attachment. Examples of such interactionsinclude a hydrophilic interaction, a hydrophobic interaction, a charged(ionic) interaction, a van de Waals interaction, or combinations of suchinteractions. In some embodiments, the immobilisation agent is passivelybound to the solid substrate. In some embodiments, the immobilisationagent is actively bound to the solid substrate.

As described herein, the use of a bound immobilisation agent providesone or more advantages to some of the embodiments of the methods and/orkits for detecting an analyte. For example, the use of a boundimmobilisation agent diminishes the potential influence ofsubstrate-reactive proteins in the sample. For example, hydrophobicproteins in the sample are less likely to affect the outcome of themethod and/or kits even if it is performed on a hydrophobic solidsubstrate, as the immobilisation agent is already bound to the solidsubstrate and the ligand on the capture agent may be specific for theimmobilisation agent.

In some embodiments, the immobilisation agent and the ligand on thecapture agent form a binding pair. A range of different immobilisationagent and ligand binding pairs may be used.

In some embodiments, the immobilisation agent and the ligand are abinding pair that is not a polyanionic-polycationic binding pair.

In some embodiments the use of an immobilisation agent-ligand bindingpair which do not bind substantially through an ionic interactionbetween a substantially polyanionic molecule and a substantiallypolycationic molecule may provide one or more advantages to the methodand/or kits for detecting an analyte in a sample. Examples of polyionicmolecules include polymeric ionic substances, or polypeptides withrepeated charged amino acids, such as a polyhistidine tag.

In some embodiments, advantages of using a immobilisation agent-ligandbinding pair that is not a polyanionic-polycationic binding pairinclude, for example, the fact that the binding between the pair ofmolecules is less dependent upon the pH of any solution contacting thebinding pair and/or the fact that the ability to reduce non-specificinteractions is difficult with polyionic binding pairs. Furthermore,many proteins present in biological milieu may specifically bind eitherpolyanions or polycations, making these components potentially difficultto detect with such an immobilisation system.

In addition, in some embodiments the use of a immobilisationagent-ligand binding pair that is not a polyanionic-polycationic bindingpair may provide other advantages including promoting the formation of acomplex between the capture agent, the analyte and the detectable agent,improving the access of such a complex to the solid substrate andpromoting the ability of the detectable agent to access the analyte fordetection purposes.

In some embodiments, the immobilisation agent and ligand binding paircomprise an anti-peptide tag antibody (for example the octapeptideDYKDDDDK (SEQ ID NO. 1) and an antibody against this peptide tag. Otherexamples of peptide tag/anti-peptide tag antibodies as binding pairs aredisclosed herein.

In some embodiments, the immobilisation agent and ligand binding pairscomprise biotin and avidin or streptavidin (or derivates thereof); metalchelate (e.g. copper, nickel, cobalt) and Histidine (e.g. histidinetagged proteins); maleic anhydride and amine (e.g. amine containingproteins); or meleimide and sulfhydryls (e.g. sulfhydryl peptides).

As described herein, in some embodiments where a peptide tag is used asa ligand, the corresponding immobilization agent may comprise an antipeptide tag antibody, as described herein.

A range of anti peptide tag antibodies may be obtained or produced by aperson skilled in the art. For example, commercially availableantibodies against the peptide tag DYKDDDDK (SEQ ID NO. 1) are describedherein. In some embodiments, the peptide tag comprisesKRITVEEALAHPYLEQYYDPTDE (SEQ ID NO. 2), a sequence derived from thecarboxy terminus of the human ERK proteins (ERK C-term peptide). Incertain embodiments, the peptide tag does not comprise a plurality ofconsecutive amino acids with the same charge.

In some embodiments, the methods and/or kits of the present disclosurecomprise an antibody capture agent and/or an antibody detectable agent.

As described herein, reference to an “antibody” is to be understood tomean an immunoglobulin molecule with the ability to bind an antigenicregion of another molecule, and includes monoclonal antibodies,polyclonal antibodies, multivalent antibodies, chimeric antibodies,multispecific antibodies, diabodies and fragments of an immunoglobulinmolecule or combinations thereof that have the ability to bind to theantigenic region of another molecule with the desired affinity includinga Fab, Fab′, F(ab′)₂, Fv, a single-chain antibody (scFv) or apolypeptide that contains at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding, such as a moleculeincluding one or more CDRs. Antibodies and/or binding fragments thereofto specific analytes may be obtained commercially or generated by knownmethods.

As described herein, in some embodiments the methods and/or kits ofpresent disclosure comprise providing the capture agent in solution.

In many previous methods for detecting an analyte using a capture agent,the capture agent is immobilised on the solid substrate prior to cominginto contact with the analyte and binding to the analyte. In someembodiments, the use of a capture agent in solution provides one or moreadvantages over the use of a capture agent immobilised on the solidsubstrate. Without being bound by theory, it is believed that the use ofthe capture agent in solution in some of the embodiments promotes thebinding of the capture agent to the analyte and thereby promotes theformation of a complex of the capture agent, the analyte and thedetectable agent.

In this regard, it will be understood that in some embodiments thecapture agent is provided in solution prior to contacting the captureagent with the sample. Accordingly, in some embodiments the captureagent is provided in a form where it is not immobilised to a solidsubstrate and is provided in a substantially liquid state.

In some embodiments, the use of an antibody capture agent in solutionalso reduces the amount of a specific antibody to a target to be reducedas compared to previous immunoassays, as more target-specific captureantibody is required in assays when the absorbed capture is pre-absorbedonto a plate. In some embodiments there is no pre-immobilisation of thecapture agent on the solid substrate.

In some embodiments, the capture agent comprises a plurality of ligandsfor the immobilisation agent. In some embodiments, the capture agentcomprises, at a plurality of sites, a ligand for the immobilisationagent. In some embodiments, the capture agent comprises differentligands. Immobilisation agent-ligand binding pairs are as describedherein.

In some embodiments, the ligand may be part of the capture agent. Forexample, the capture agent may comprise poly-histidine residues, aminegroups or sulfhydryl groups that are able to bind to the immobilisationagent.

In some embodiments, the ligand is bound to the capture agent.

In some embodiments, the ligand for the immobilisation agent comprises apeptide tag, such as the octapeptide DYKDDDDK (SEQ ID NO. 1), sometimesreferred to as FLAG-tag.

As described herein, peptide-tags are polypeptide tags that can beconjugated to an another agent, such as a protein or an antibody, oradded to a protein using recombinant DNA technology. As describe herein,one example of a peptide tag is the peptide DYKDDDDK (SEQ ID NO. 1),which can be used in different assays that utilize recognition by anantibody. Other examples of peptide tags are described herein.

Adding a peptide tag to a protein allows the protein to be bound and/orimmobilised by an antibody against the peptide tag sequence. In someembodiments, a peptide tag may also be used in conjunction with otheraffinity tags for example a polyhistidine tag (His-tag), HA-tag ormyc-tag.

The addition of a peptide tag to the capture agent to form a conjugatemay be achieved by a suitable known method.

As described herein, in some embodiments the methods and/or kits fordetecting an analyte comprise providing a detectable agent which canbind to the analyte.

In some embodiments the detectable agent is provided in solution.Accordingly, in some embodiments the detectable agent is provided in asubstantially liquid state.

In some embodiments, the use of a detectable agent in solution mayprovide one or more advantages to the methods and/or kits of the presentdisclosure, including promoting the formation and detection of a complexof the capture agent, the analyte and the detectable agent.

In some embodiments, the methods and/or kits of the present disclosureprovide one or more detectable agents, the one or more detectable agentsbeing able to bind to one or more analytes to be detected.

In some embodiments, the detectable agent comprises an antibody(including a binding fragment thereof), an aptamer, or a proteinreceptor or ligand (or binding fragment thereof) as described herein.Examples of antibodies and binding fragments thereof are as describedherein.

Protein receptors or ligands used as a detectable agent may comprise thewhole receptor or ligand or a fragment thereof (for example a fragmentcomprising a binding domain of the receptor or ligand). In someembodiments, the receptor or ligand (or fragment thereof) may comprise afusion protein. Fusion partners may include, for example, fluorescentfusion partners (e.g. GFP) and immunoglobulin fusion partners. Fusionpartners and methods for preparing fusion proteins are known in the art.In some embodiments, the fusion partner may act to stabilise thereceptor or ligand (or fragment thereof), provide a detectable signal(e.g. for fluorescent fusion partners) or provide a target for antibody,or other, binding or immobilisation.

Aptamers used as a detectable agent may be obtained commercially orgenerated by known methods.

In some embodiments, the detectable agent may comprise a detectable tag.In some embodiments, the detectable tag may be applied to the detectableagent (for example bound to the detectable agent) or may be part of thedetectable agent (for example the detectable agent may include thedetectable tag as a fusion partner, a labelled amino acid or labellednucleotide).

Examples of suitable detectable tags include antigens, enzymes,fluorophores, quenchers, radioactive isotopes and luminescent labels. Itwill be appreciated that the detectable tag may be detected directly orindirectly via a further molecule that can produce a detectable signal.

Antigens that may be used as a detectable tag may include, for example,any antigenic component of the detectable agent that may be targeted bya secondary detectable agent. For example, in some embodiments, asecondary antibody may be used to detect an antigen on a detectableagent. The secondary antibody may be fluorescently or enzymaticallylabelled. In embodiments where the detectable agent is a primaryantibody (ie. an analyte binding antibody), the secondary antibody mayhave binding affinity to an antigen on the primary antibody. Forexample, the antigen may be derived from the host in which thedetectable agent was raised.

In some embodiments, the detectable tag comprises an enzyme. Enzymesthat may be used as detectable tags include, for example, enzymes thatresult in the conversion of a substrate into a detectable product(generally resulting in a change in colour or fluorescence or generationof an electrochemical signal). Such enzymes may include, for example,horseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, acetylcholinesterase, luciferase, catalase orcombinations thereof. Depending on the enzyme and substrate used,detection may be performed with a spectrophotometer, fluorometer,luminometer, electrochemical detection means. Other detection means arecontemplated.

Radioactive isotopes that may be used as detectable tags include, forexample, ³H, ¹⁴C, ³²P, ³⁵S, or ¹³¹I. Other isotopes are contemplated.The radioisotope may be conjugated to a detectable agent or incorporatedinto a detectable agent by translation of mRNA encoding the detectableagent in the presence of radiolabelled amino acids. Radioisotopes andmethods for conjugating radioactive isotopes to molecules such asproteins are known in the art. Radioisotopes may be detected usinggamma, beta or scintillation counters.

Fluorophores that may be used as detectable tags include, for example,resorufin, fluorescein (fluorescein isothiocyanate, FITC), rhodamine(tetramethyl rhodamine isothiocyanate, TRITC), green fluorescent protein(GFP), phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrinand phycoerythrocyanin, derivatives of any of the foregoing) orcombinations thereof. In some embodiments, the detectable tag may bepart of the detectable agent (e.g. in the form of a fusion protein or aprotein comprising fluorescent amino acids). Fluorophores may besubjected to applied stimulation (for example light of a suitableexcitation wavelength) to promote fluorescence.

Luminescent compounds that may be used as detectable tags include, forexample, chemiluminescent and/or bioluminescent compounds. Thesecompounds may be used to label the detectable agent. The presence of thechemiluminescent-tag may be determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of useful chemiluminescent labelling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester or combinations thereof. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent antibody is determined by detecting thepresence of luminescence. Examples of bioluminescent compounds includeluciferin, luciferase and aequorin.

In some embodiments, the methods and/or kits for detecting an analytecomprise contacting the sample, the capture agent, the detectable agentand the solid substrate, in a reaction vessel, to form a mixture. Insome embodiments, the components are brought into contact with eachother, in the reaction vessel, to allow the formation of a complexbetween the capture agent, the analyte and the detectable agent, thecomplex being able to be immobilised on the solid substrate (via theligand on the solid substrate) concurrently or after its formation. Asdescribed herein, this may provide advantages to the performance ofcertain methods and/or kits of the present disclosure.

As described herein, in some embodiments the methods and/or kits of thepresent disclosure comprise contacting the sample, the capture agent,the detectable agent and the solid to allow binding of the capture agentand the detectable agent to the analyte to form a complex. Uponformation, the complex may be immobilized on the solid substrate via theligand binding to the immobilisation agent bound to the solid substrate.

As described herein, in some embodiments, the methods and/or kitsprovide contacting in one or more of at least two reaction vessels, oneor more samples, one or more capture agents and one or more detectableagents to allow the formation of one or more complexes comprising ananalyte, a capture agent and a detectable agent.

In some embodiments, the methods and/or kits provide contacting one ormore complexes with the solid substrate, such that the immobilisationagent may bind the one or more complexes via the ligand.

In some embodiments, prior to bringing the components into contact witheach other in the reaction vessel, specific individual components may bebrought into contact prior with each other.

In some embodiments, contacting of one or more of the individualcomponents may occur in the reaction vessel or may occur in one or moreseparate reaction vessels.

In some embodiments, the sample, the capture agent, the detectable agentand the solid substrate are not contacted in a separate vessel prior tocontacting in the reaction vessel. Thus, the combination of thecomponents is contacted together for the first time in the reactionvessel.

In some embodiments, the sample and the solid substrate are contactedprior to contacting with the capture agent and/or the detectable agent.As discussed herein, this particular method of contacting provides oneor more advantages to the performance of some embodiments of the methodsand/or kits, as the capture agent and/or the detectable agent areexposed to the analyte in the presence of the solid substrate.

In some embodiments, the sample (and analyte(s) therein) is exposed tothe solid substrate prior to exposure to either or both of the captureagent and the detectable agent. As discussed herein, in some embodimentsthis particular method of contacting provides an advantage to theperformance of the method, as the capture agent and/or the detectableagent do not come into contact with the analyte until the analyte is inthe presence of the solid substrate. For example, without being bound bytheory, this may provide advantages to the formation of the complexbetween the capture agent, the analyte and the detectable agent.

In some embodiments, the sample and the solid substrate are contacted inthe reaction vessel.

In some embodiments where the solid substrate forms part of the reactionvessel, the sample may be added to the reaction vessel and subsequentlythe capture agent and/or the detectable agent are brought into contactwith the sample and the solid substrate.

In some embodiments, the sample and the solid substrate are notsubstantially incubated prior to contacting with the capture agentand/or the detectable agent. In some embodiments, this may provide anadvantage by reducing the time required to detect the analyte.

In some embodiments, the capture agent and the detectable agent arebrought into contact with each other before they are contacted witheither or both of the sample and the solid substrate. Typically, thismay be achieved by first contacting the capture antibody and thedetectable agent in a separate vessel.

In some embodiments wherein the solid substrate forms part of thereaction vessel, the capture agent and the detectable agent are broughtinto contact with each other and then placed in the reaction vesselcontaining a sample.

In some embodiments, the capture agent and the detectable agent aresequentially contacted with the previously contacted sample and/or thesolid substrate.

In some embodiments, the sample and the solid substrate are brought intocontact and then each of the capture agent and the detectable agent arethen brought into contact with the sample and the solid substrate. Insome embodiments, the capture agent is first contacted with the sampleand the solid substrate and subsequently the detectable agent is broughtinto contact with the capture agent, the sample and the solid substrate.In some embodiments, the detectable agent is first contacted with thesample and the solid substrate and subsequently the capture agent isbrought into contact with the detectable agent, the sample and the solidsubstrate.

As described herein, in some embodiments the complex comprising theanalyte, capture agent and detectable agent is formed prior to bindingbetween the immobilisation agent and the ligand. The complex may beformed by sequential or concurrent addition of the capture agent anddetectable agent to the analyte prior to contacting the complex with theimmobilisation agent on the solid substrate.

In some embodiments, there is an incubation of the contacted sample, thesolid substrate, the capture agent and the detectable agent.

In some embodiments, there is an incubation of the complex and the solidsubstrate.

In some embodiments, there is an incubation of the contacted sample, thesolid substrate, the capture agent and the detectable agent prior towashing of the solid substrate.

In some embodiments, there is an incubation of the complex and the solidsubstrate prior to washing of the solid substrate.

In some embodiments, the incubating is 2 hours or less, 90 minutes orless, 80 minutes or less, 70 minutes or less, 1 hour or less, 50 minutesor less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15minutes or less, 10 minutes or less, or 5 minutes or less. In someembodiments, if desired, there will be no incubation.

In some embodiments, the incubation is between 10 minutes to 2 hours, 10minutes to 1 hour, 15 minutes to 2 hours, 15 minutes to 1 hour, 30minutes to 2 hours, 30 minutes to 1 hour, or 1 hour to 2 hours. In someembodiments, the incubation is at least 5, 10, 15, 20, 25, 30, 60, 70,80, 90 or 120 minutes.

In some embodiments, the incubating is 30 minutes or less. In someembodiments, the incubating is 25 minutes or less, 15 minutes or less,10 minutes or less or 5 minutes or less. In some embodiments, theincubating is 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 5 to 15minutes, 10 to 30 minutes, 10 to 25 minutes, 10 to 15 minutes, 15 to 30minutes, 15 to 25 minutes, 15 to 20 minutes, 20 to 30 minutes, or 20 to25 minutes. In some embodiments, the incubating is at least 5, 10, 15,20, 25, or 30 minutes.

In some embodiments, the incubation of the contacted sample, the solidsubstrate, the capture agent and the detectable agent prior to washingof the solid substrate occurs in the same reaction vessel. In someembodiments the incubation of the contacted sample, the solid substrate,the capture agent and the detectable agent prior to washing of the solidsubstrate occurs in a separate reaction vessel.

In some embodiments, the incubation of the complex and the solidsubstrate prior to washing of the solid substrate occurs in the samereaction vessel. In some embodiments the incubation of the complex andthe solid substrate prior to washing of the solid substrate occurs in aseparate reaction vessel.

As described herein, in some embodiments the methods and/or kitscomprise washing the solid substrate one or more times to remove boththe capture agent and the detectable agent not bound to the solidsubstrate via the ligand. The washing of the solid substrate may beperformed using a suitable method, sufficient to remove capture agentand detectable agent not bound to the solid substrate via the ligand onthe capture agent.

Washing the solid substrate prior to detection of the detectable agentallows the removal of unbound detectable agent and/or detectable agentnot bound via the capture agent, which can decrease the level ofbackground signal and hence improve sensitivity. Methods for washingsteps are known and generally involve repeated addition and removal ofbuffer.

The solid substrate may be washed one or more times, and with one ormore buffers. In some embodiments, the solid substrate may be washed twoor more times, and with one or more buffers. In some embodiments, thesolid substrate may be washed three or more times, and with one or morebuffers.

In some embodiments, there is no additional washing of the solidsubstrate after contacting of the solid substrate with any one or moreof the sample, the capture agent and the detectable agent.

For example, in embodiments utilising a peptide/antibody capture systemor a steptavidin/biotin capture system, such systems may assist inreducing the steps involving washing of the solid substrate.

In these embodiments it will be appreciated that there may be noadditional washing of the solid substrate after contacting of the solidsubstrate with any one or more of the sample, the capture agent and thedetectable agent. However, in some embodiments, if desired, additionalwashing of the solid substrate after contacting of the solid substratewith any one or more of the sample, the capture agent and the detectableagent may be conducted. In some embodiments, one, two, three or fourwashings of the solid substrate after contacting of the solid substratewith any one or more of the sample, the capture agent and the detectableagent may be conducted.

In some embodiments, the methods and/or kits may be performed using onlya single/one wash step conducted during the entire method. In someembodiments, the use of a one wash protocol may provide one or moreadvantages. For example, not only does the use of a one wash protocolprovide advantages to the number of handling steps involved and the timerequired to conduct the method, the one wash protocol may also providean improvement in the efficiency and performance of the method. It willbe appreciated that in a one wash protocol, the solid substrate may bewashed one or more times at that step in the protocol.

However, in certain embodiments, the one wash protocol may be varied, ifdesired, to add additional washes or rinses at various stages of theprotocol or to add additional washes or rinses at various stages of theprotocol.

In some embodiments, there is no additional washing of the solidsubstrate after contacting of the solid substrate with any one or moreof the sample, the capture agent and the detectable agent.

As described previously herein, in some embodiments the methods and/orkits of the present disclosure comprise detecting the analyte bydetecting the presence of the detectable agent bound to the solidsubstrate.

In this regard, in some embodiments the detection of the analyte isachieved by detecting the presence of the detectable agent present inthe complex immobilised to the solid substrate in the reaction vessel.

In some embodiments the methods and/or kits of the present disclosurecomprise detecting the analyte by detecting the presence of thedetectable agent present in the complex immobilised to the solidsubstrate in the reaction vessel.

In some embodiments, the methods and/or comprise detecting the presenceof one or more immobilised complexes on the solid substrate by detectionof the one or more detectable agents.

The detection of an analyte by detecting the presence of the detectableagent bound to the substrate may be achieved by a suitable methodspecific to the detectable agent. Examples of detectable agents are asdescribed herein.

As described herein, the time taken to perform a method for detecting ananalyte in a sample is an important consideration.

For example, ELISA can be time-intensive and it is not uncommon in ELISAfor incubation steps to be performed after the addition of eachindividual component of the ELISA.

As discussed herein, in some embodiments the present disclosureminimises the number of incubation, handling and/or washing steps. Insome embodiments, this makes the method of the present disclosureparticularly amenable to automation. Previous assays are difficult toautomate as multiple handling steps are needed, including severalaspiration, dispensing, and washing steps.

In some embodiments, reducing the number of incubation steps and/orwashing steps that are required may allow the duration of the complex tosolid substrate binding step to be maximised without increasing thetotal duration of the method. In some embodiments, increasing theduration of the complex to solid substrate binding step may increase thesensitivity of the method. Some embodiments of the present disclosurecontemplate, if desired, various combinations as to the number ofincubation, handling and/or washing steps.

In some embodiments, the detection of the analyte is achieved in a timeof 2 hours or less from contacting the sample with the capture agentand/or the detectable agent.

In some embodiments, the detection of the analyte is achieved in a timeof 90 minutes or less, 70 minutes or less, 60 minutes or less, 50minutes or less, 45 minutes or less, 40 minutes or less, 30 minutes orless, 20 minutes or less, 15 minutes or less, or 10 minutes or less fromcontacting the sample with the capture agent and/or the detectableagent. In some embodiments, the detection of the analyte is achieved ina time period of between 10 minutes to 120 minutes, 10 minutes to 90minutes, 10 minutes to 60 minutes, 15 minutes to 120 minutes, 15 minutesto 90 minutes, 15 minutes to 60 minutes, 15 minutes to 30 minutes, 30minutes to 120 minutes, 30 minutes to 90 minutes, 30 minutes to 60minutes, 45 minutes to 120 minutes, 45 to 90 minutes, or 45 minutes to60 minutes from contacting the sample with the capture agent and/or thedetectable agent.

In some embodiments, the detection of the analyte is achieved in a timeof less than 60 minutes from contacting the sample with the captureagent and/or the detectable agent. In some embodiments, the detection ofthe analyte is achieved in a time of less than 30 minutes fromcontacting the sample with the capture agent and/or the detectableagent. In some embodiments, the detection of the analyte is achieved ina time of less than 15 minutes from contacting the sample with thecapture agent and/or the detectable agent. In some embodiments, thedetection of the analyte is achieved in a time of less than 10 minutesfrom contacting the sample with the capture agent and/or the detectableagent.

As described herein, in some embodiments the use of a capture system mayassist in reducing the time required to undertake an assay. For example,in embodiments utilising a peptide/antibody capture system or asteptavidin/biotin capture system, such systems may assist in reducingthe time to undertake such an assay.

As described herein, in some embodiments the use of a capture system mayassist in reducing the washing of the solid substrate and/or reducingthe time required to undertake an assay. For example, in embodimentsutilising a peptide/antibody capture system or a steptavidin/biotincapture system, such systems may assist in reducing the washing of thesolid substrate and in reducing the time to undertake such an assay.

As described herein, as some embodiments of the methods also allowdetectable signals to be produced with less capture agent and/or reducedsolid substrate surface area relative to ELISA, some methods may besuitable for microfluidic systems, where miniaturisation of structuresand minimisation of reagents used is desirable. Accordingly, in someembodiments, the method may be performed in a microfluidic system, asdescribed herein.

In some embodiments, the methods and/or kits show a low variability fordetecting an analyte between reactions. In some embodiments, the methodsand/or kits show a low intra-plate variability. In certain embodiments,the variability is 30% or less, 20% or less, or 10% or less. Forexample, in certain embodiments the methods and/or kits shows a lowintra-plate variability for detecting an analyte, such as an intra-platevariability of 30% or less, 20% or less, or 10% or less.

In some embodiments, the methods and/or kits of the present disclosuremay also be performed by utilising reagents and/or instructions.

In some embodiments, a further advantage of some embodiments of themethods and/or kits of the present disclosure is the ability to use asingle assay plate or platform that is suitable for many different assaykits. This may provide manufacturers with a number of benefits,including reduced cost, labor and quality control requirements, incomparison to preparing a different assay plate for every assay kit, asis the current standard for ELISA kit manufacture. In addition, in someembodiments inputs can be reduced by the ability to use less of thetarget-specific capture antibody, again reducing costs and qualitycontrol requirements, as single batches of target-specific antibodiescan be used for more assay kits.

In some embodiments, the present disclosure provides a method fordetecting one or more analytes in one or more samples using a singleassay platform, the method comprising:

-   -   providing one or more samples comprising one or more analytes to        be detected;    -   providing a single assay platform comprising at least two        reaction vessels, the at least two reaction vessels comprising a        solid substrate comprising a bound immobilisation agent;    -   providing one or more capture agents, the one or more capture        agents being able to bind to the one or more analytes to be        detected and comprising, at a plurality sites, a ligand for the        immobilisation agent;    -   providing one or more detectable agents, the one or more        detectable agents being able to bind to the one or more analytes        to be detected;    -   contacting in one or more of the at least two reaction vessels        in the assay platform, the one or more samples, the one or more        capture agents and the one or more detectable agents to allow        the formation of one or more complexes comprising an analyte, a        capture agent and a detectable agent;    -   contacting the one or more complexes with the solid substrate        such that the immobilisation agent may bind the one or more        complexes via the ligand; and    -   detecting the presence of one or more immobilised complexes on        the solid substrate by detection of the one or more detectable        agents.

In some embodiments, the bound immobilisation agent is the sameimmobilisation agent.

In some embodiments, the present disclosure provides a method fordetecting one or more analytes in one or more samples using a singleassay platform, the method comprising:

-   -   providing one or more samples comprising one or more analytes to        be detected;    -   providing a single assay platform comprising at least two        reaction vessels, the at least two reaction vessels comprising a        solid substrate comprising the same bound immobilisation agent;    -   providing one or more capture agents, the one or more capture        agents being able to bind to the one or more analytes to be        detected and comprising, at a plurality sites, a ligand for the        immobilisation agent;    -   providing one or more detectable agents, the one or more        detectable agents being able to bind to the one or more analytes        to be detected;    -   contacting in one or more of the at least two reaction vessels        in the assay platform, the one or more samples, the one or more        capture agents and the one or more detectable agents to allow        the formation of one or more complexes comprising an analyte, a        capture agent and a detectable agent;    -   contacting the one or more complexes with the solid substrate        such that the immobilisation agent may bind the one or more        complexes via the ligand; and    -   detecting the presence of one or more immobilised complexes on        the solid substrate by detection of the one or more detectable        agents.

In some embodiments, a kit is utilised for performing the methods of thepresent disclosure. The kit may comprise one or more of the reagentsand/or one or more components herein described and/or instructions toassist in the performance of the method.

In some embodiments, the present disclosure provides a kit for detectingan analyte in a sample, the kit comprising:

-   -   an assay platform comprising a plurality of reaction vessels,        one or more of the reaction vessels comprising a bound        immobilisation agent;    -   a capture agent which can bind to an analyte, wherein the        capture agent comprises, at a plurality of sites, a ligand for        the immobilisation agent;    -   an antibody detectable agent which can bind to the analyte,        wherein the antibody detectable agent comprises a detectable        tag; and    -   instructions for detecting the analyte.

In some embodiments, the kit comprises instructions for detecting theanalyte in a time of 2 hours or less, 90 minutes or less, 80 minutes orless, 70 minutes or less, 1 hour or less, 45 minutes or less, 30 minutesor less, 15 minutes or less, or 10 minutes or less.

In some embodiments, the kit comprises instructions for detecting theanalyte comprise instructions for detecting the analyte in a time of 2hours or less, 90 minutes or less, 80 minutes or less, 70 minutes orless, 1 hour or less, 45 minutes or less, 30 minutes or less, 15 minutesor less, or 10 minutes or less from contacting the sample with thecapture agent and/or the detectable agent.

In some embodiments, the kit comprises instructions for the detecting ofthe analyte in a time period of between 10 minutes to 120 minutes, 10minutes to 90 minutes, 10 minutes to 60 minutes, 10 minutes to 30minutes, 15 minutes to 120 minutes, 15 minutes to 90 minutes, 15 minutesto 60 minutes, 30 minutes to 120 minutes, 30 minutes to 90 minutes, 30minutes to 60 minutes, 45 minutes to 120 minutes, 45 to 90 minutes, 45minutes to 75 minutes, or 45 minutes to 60 minutes.

In some embodiments, the kit comprises instructions for the detecting ofthe analyte in a time period of between 10 minutes to 120 minutes, 10minutes to 90 minutes, 10 minutes to 60 minutes, 10 minutes to 30minutes, 15 minutes to 120 minutes, 15 minutes to 90 minutes, 15 minutesto 60 minutes, 30 minutes to 120 minutes, 30 minutes to 90 minutes, 30minutes to 60 minutes, 45 minutes to 120 minutes, 45 to 90 minutes, 45minutes to 75 minutes, or 45 minutes to 60 minutes from contacting thesample with the capture agent and/or the detectable agent.

In some embodiments, the immobilisation agent is avidin, streptavidinand/or a derivative thereof and the ligand is biotin and/or a derivativethereof.

In some embodiments, the immobilisation agent is an anti peptide tagantibody and the ligand is a peptide tag. Other immobilisation agent andligand binding pairs are as described herein.

In some embodiments, the capture agent comprises an antibody or afragment thereof. In some embodiments, the capture agent comprises anantibody capture agent. In some embodiments, capture agent is insolution. In some embodiments, the antibody capture agent is insolution. Capture agents are as described herein.

In some embodiments, the detectable agent comprises an antibody or afragment thereof. In some embodiments, the detectable agent comprises anantibody detectable agent. In some embodiments, the detectable agent isin solution. In some embodiments, the antibody detectable agent is insolution. Detectable agents are as described herein.

As described herein, in some embodiments, a further advantage of someembodiments of the methods and/or kits of the present disclosure is theability to use a single assay plate or platform that is suitable formany different assay kits. In some embodiments, this may providemanufacturers with a number of benefits, including reduced cost, laborand quality control requirements, in comparison to preparing a differentassay plate for every assay kit, as is the current standard for ELISAkit manufacture. In addition, in some embodiments inputs can be reducedby the ability to use less of a target-specific capture antibody, againreducing costs and quality control requirements, as single batches oftarget-specific antibodies can be used for more assay kits.

In some embodiments, the kit comprises an assay platform. In someembodiments, the assay platform comprises a plurality of reactionvessels. In some embodiments, one or more of the reaction vesselscomprises a bound immobilisation agent. In some embodiments, one or moreof the reaction vessels comprise a solid substrate. In some embodiments,the assay platform comprises a multi-well plate, such as a microtitreplate, and the one or more reaction vessels comprise a well of themulti-well plate.

In some embodiments, the instructions comprise instructions forutilising only a single wash of the solid substrate after contacting ofthe solid substrate with any one or more of the sample, the captureagent and the detectable agent.

The present disclosure is further described by the following examples.It is to be understood that the following description is for the purposeof describing particular embodiments only and is not intended to belimiting with respect to the above description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows for the purposes of comparison, three ELISA protocols forthe detection of phosphorylated ERK 1/2 (pERK) were examined, usingvarious concentrations of a cellular lysate containing pERK. (1) Asimultaneous ELISA format, whereby the assay components, namely thecapture antibody (anti-pERK-biotin), the analyte (cellular lysate), andthe detection antibody (anti-ERK-HRP), were incubated concurrently in astreptavidin-coated microplate. (2) A standard multi-incubation ELISAformat, whereby the capture antibody was first incubated in astreptavidin-coated microplate, followed by the analyte, and finally thedetection antibody. (3) A standard multi-incubation ELISA format,whereby the analyte was incubated in a capture-antibody coatedmicroplate, followed by a detection antibody. The assays were incubatedfor either 30 min (FIG. 1A) for each incubation step or 60 min (FIG. 1B)for each incubation step, and the wells were subjected to a standardwash cycle between each incubation step for each assay. After the finalincubation and wash, QuantaRed™ HRP substrate was added to the wells,and each plate was incubated for 10 min in the dark. The fluorescentsignal in the wells was measured at 550ex/600em nm. FIGS. 1A and 1B showthe mean and standard deviations for the duplicate data points at eachpERK lysate concentration analyzed. In this Figure the comparisonclearly demonstrate comparable assay performance over a shorter timeperiod when the assay components are incubated concurrently, comparedwith standard ELISA protocols whereby assay components are incubatedsequentially.

FIG. 2 shows for the purposes of comparison, three ELISA protocols forthe detection of phosphorylated ERK 1/2 (pERK), using variousconcentrations of a cellular lysate containing pERK. (1) A simultaneousELISA format, whereby the assay components, namely the capture antibody(anti-pERK-biotin), the analyte (cellular lysate), and the detectionantibody (anti-ERK-HRP), were incubated concurrently in astreptavidin-coated microplate for either 30 min (FIG. 2A) or 60 min(FIG. 2B). (2) A standard multi-incubation ELISA format, whereby thecapture antibody was first incubated in a streptavidin-coated microplatefor 10 min, followed by the analyte for 10 min, and finally thedetection antibody for 10 min, giving a total cumulative assayincubation time of 30 min (FIG. 2A). (3) A standard multi-incubationELISA format, whereby the analyte was incubated in a capture-antibodycoated microplate for 30 min, followed by the detection antibody for 30min, giving a total cumulative assay time of 60 min (FIG. 1B). The wellswere subjected to a standard wash cycle between each incubation step foreach assay. After the final incubation and wash, QuantaRed™ HRPsubstrate was added to the wells, and each plate was incubated for 10min in the dark. The fluorescent signal in the wells was measured at550ex/600em nm. FIGS. 2A and 2B show the mean and standard deviationsfor the duplicate data points at each pERK lysate concentrationanalyzed. In this Figure, the comparison clearly demonstrate betterassay performance the same total assay time period when the assaycomponents are incubated concurrently, compared with standard ELISAprotocols whereby assay components are incubated sequentially.

FIG. 3 shows for the purposes of comparison, the concentration of thecapture antibody (anti-phospho-ERK) required for optimal assayperformance for three ELISA protocols for the detection ofphosphorylated ERK 1/2, using varying concentrations of the captureantibody in combination with a fixed concentration of both cellularlysate, and detection antibody. (1) A simultaneous ELISA format, wherebythe assay components, namely the capture antibody (anti-pERK-biotin),the analyte (cellular lysate), and the detection antibody(anti-ERK-HRP), were incubated concurrently in a streptavidin-coatedmicroplate for 120 min. (2) A simultaneous ELISA format, whereby theassay components, namely the capture antibody (anti-pERK-peptide), theanalyte (cellular lysate), and the detection antibody (anti-ERK-HRP),were incubated concurrently in an anti-peptide antibody-coatedmicroplate for 120 min. (3) A standard multi-incubation ELISA format,whereby the analyte was incubated in a capture-antibody(non-biotinylated) coated microplate for 120 min, followed by thedetection antibody for 120 min. The wells were subjected to a standardwash cycle between each incubation step for each assay. After the washcycle, HRP substrate was added to the wells, and the plates wereincubated for 10 min in the dark. The fluorescent signal in the wellswas measured at 540ex/590em nm. FIG. 3 shows the mean and standarddeviations for the duplicate data points for each target analyzed. Inthis Figure, the comparison clearly demonstrates that optimal assayperformance is achieved with lower capture antibody concentrations whenthe assay components are incubated concurrently for both biotin-captureand peptide-capture protocols, when compared with standard ELISAprotocols whereby analytes are incubated sequentially, and washedbetween incubations. This data demonstrates that the assay has thepotential to lower input costs for ELISA plate manufacture.

FIG. 4 shows for the purposes of comparison, the requirement forsequential incubations for optimal assay performance for two ELISAprotocols for the detection of phosphorylated ERK 1/2. (1) Asimultaneous ELISA format, whereby the assay components, namely thecapture antibody (anti-phospho-ERK-peptide), the analyte (cellularlysate), and the detection antibody (anti-ERK-HRP), were incubatedconcurrently in an antipeptide antibody-coated microplate for 120 min.(2) A sequential ELISA format, whereby the solution-phase assaycomponents, namely the capture antibody (anti-pERK-peptide), the analyte(cellular lysate), and the detection antibody (anti-ERK-HRP), wereincubated concurrently in a separate reaction vessel for 60 min. Theassay components were subsequently transferred to an antipeptideantibody-coated microplate for 60 min. At the conclusion of incubationon the antipeptide antibody-coated assay microplate, both protocolsrequired a standard wash cycle. After the wash cycle, HRP substrate wasadded to the wells, and the plates were incubated for 10 min in thedark. The fluorescent signal in the wells was measured at 540ex/590emnm. FIG. 4 shows the mean and standard deviations for the duplicate datapoints for each target analyzed. In this Figure, the comparison clearlydemonstrates that no benefit to assay performance is achieved with theinclusion of a pre-incubation step prior to introduction to a solidsubstrate carrying the immobilization agent.

FIG. 5 shows a single-incubation, single-wash ELISA, was performed usinga 3-antibody configuration. The assay components, namely the captureantibody (anti-pERK-biotin), the analyte (cellular lysate), thedetection antibody (rabbit anti-ERK), and a generic anti-rabbit-HRPantibody, were incubated concurrently in a streptavidin-coatedmicroplate for 120 min (signal), and compared with a similar assay runwith a buffer-only control for the analyte (noise). The wells weresubjected to a standard wash cycle after the incubation step, andSigmaFAST™ HRP substrate was added to the wells, and each plate wasincubated for 10 min in the dark. The colorimetric signal in the wellswas measured at 450 nm. FIG. 5 shows the mean and standard deviationsfor the duplicate data points at each pERK lysate concentrationanalyzed. In this Figure, the assay clearly demonstrates the utilitywhereby the assay components are incubated concurrently.

FIG. 6 shows the detection of different kinases by a single incubation,single wash ELISA. Cell lysates containing either phosphorylated S6p240/44, AKT pT308 or AKT pS473 (signal), or buffer-only controls(noise) were added to separate wells of an assay microplate(streptavidin coated 384-well Nunc Maxisorp™ plate). The reaction wasstarted by the addition of target-specific antibody pairs (onebiotinylated and the other conjugated to HRP) to the lysates. The assayswere incubated for 2 h, then subjected to a wash cycle. After the washcycle, QuantaRed™ HRP substrate was added to the wells, and the platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 550ex/600em nm. FIG. 6 shows the mean and standarddeviations for the duplicate data points for each target analyzed. Inthis Figure, the assay clearly demonstrates efficacy for severaldifferent targets, whereby the assay components are incubatedconcurrently.

FIG. 7 is a schematic diagram showing a microfluidic cartridge suitablefor use in accordance with some embodiments of the methods of thepresent disclosure.

FIG. 8 demonstrates the results of electrochemical detection of pERK ina microfluidic system. FIG. 8A shows the raw results of electrochemicaldetection (in mV) during the substrate flow through phase and substrateincubation phase. FIG. 8B shows a pERK standard curve generated usingdata taken from 60 seconds after injection of substrate (during flowthrough phase). FIG. 8C shows a pERK standard curve generated using datataken from 180 seconds after injection of substrate (at the end of thesubstrate incubation phase).

FIG. 9 shows the results of electrochemical detection of pAKT473 in amicrofluidic system. FIG. 9A shows the raw results of electrochemicaldetection (in mV) during the substrate flow through phase and substrateincubation phase. FIG. 9B shows a pAKT473 standard curve generated usingdata taken from 60 seconds after injection of substrate (during flowthrough phase). FIG. 9C shows a pAKT473 standard curve generated usingdata taken from 180 seconds after injection of substrate (at the end ofthe substrate incubation phase).

FIG. 10 demonstrates equivalent assay performance with variouspermutations on the order of delivery of assay components to the assaywell, using a peptide capture antibody conjugate (anti-pERK-peptide)(FIG. 10A), or biotin capture antibody conjugate (anti-pERK-biotin)(FIG. 10B), as the assay capture reagent The assay components were addedin various permutations (refer to example 7, Tables 1 and 2). Individualassay components were added 1 min apart to the plates and incubated for2 h at room temperature, then subjected to a wash cycle. After the washcycle, HRP substrate was added to the wells, and the plates wereincubated for 10 min in the dark. The fluorescent signal in the wellswas measured at 540ex/590em nm. FIG. 10 shows the mean and standarddeviations for the duplicate data points for each target analyzed. InFIG. 10, the assays clearly demonstrate that when added within the shorttime period described, the order of addition of individual assaycomponents does not affect assay performance, compared with assaycomponents that are added simultaneously.

FIG. 11 shows detection of recombinant human EGF, IL-2 and TNFα, ineither PBS/0.5% BSA or human serum. Peptide-capture antibody conjugates,and HRP-detection antibody conjugates specific for each of EGF (FIG.11A), IL-2 (FIG. 11B) and TNFα (FIG. 11C) were prepared. RecombinantEGF, IL-2 or TNFα were prepared at concentrations ranging from 100 ng/mLto 10 fg/mL, in either PBS/0.5% BSA, or human serum, and 50 μL/well ofeach analyte was added to an ELISA assay plate coated with ananti-peptide antibody. The assays were initiated by addition of mixturescontaining both specific antibodies for each of EGF, IL-2 or TNFα, alongwith a general anti-HAMA composition available commercially fromBioreclamation LLS (Westbury, N.Y., USA—‘Immunoglobulin InhibitingReagent (IIR)), to the appropriate ELISA plate wells. The assays wereincubated for 1 h, then subjected to a wash cycle. After the wash cycle,HRP substrate was added to the wells, and the plates were incubated for10 min in the dark. The fluorescent signal in the wells was measured at540ex/590em nm. FIG. 11 shows the mean and standard deviations for theduplicate data points for each target analyzed. In this Figure, theassay clearly demonstrates efficacy for several different targets inserum, whereby the assay components are incubated concurrently. The highsignal for EGF in human serum is due to the presence of endogenous EGFprotein(s) in this medium.

FIG. 12 shows detection of recombinant human EGF, IL-2 and TNFα in a 15min total assay time. Peptide-capture antibody conjugates, andHRP-detection antibody conjugates specific for each of EGF (●), IL-2 (▪)and TNFα (▴) were prepared. Recombinant EGF, IL-2 or TNFα were preparedat concentrations ranging from 100 ng/mL to 10 fg/mL, in PBS/0.5% BSA,and 50 μL/well of each analyte was added to an ELISA assay plate coatedwith an anti-peptide antibody. The assays were initiated by addition ofmixtures containing both specific antibodies for each of EGF, IL-2 orTNFα to the appropriate ELISA plate wells. The assays were incubated for10 min, then subjected to a wash cycle. After the wash cycle, HRPsubstrate was added to the wells, and the plates were incubated for 5min in the dark. The fluorescent signal in the wells was measured at540ex/590em nm. FIG. 12 shows the mean and standard deviations for theduplicate data points for each target analyzed. In this Figure, theassay clearly demonstrates efficient detection within 15 min total assaytime for several different targets, using certain embodiments, wherebythe assay components are incubated concurrently.

FIG. 13 shows intra-plate variation observed for 2 separatesingle-incubation ELISAs for either phospho-AKT (pSer473) orphospho-STAT3.

FIG. 14 shows detection of TNFα in tissue culture supernates.

FIG. 15 shows detection of either phospho-AKT (pSer473) or phospho-ERKin a 25 min total assay time. For each target, recombinant active (FIG.15A) phospho-AKT or phospho-ERK (FIG. 15B) was diluted as indicated, tovarious concentrations using 1× Lysis buffer containing 0.1% BSA andadded to 4 replicate wells of a 96-well streptavidin-coated microplate.To initiate the assay reaction, for either target, a mixture of thebiotin-conjugated capture antibody, and the HRP-conjugated detectionantibody were added to the lysates, and incubated for 1 hour. The wellswere subjected to a standard wash cycle for each assay. After the washcycle, QuantaRed™ HRP substrate was added to the wells, and each platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 550ex/600em nm. FIGS. 15A and 15B show the datapoints at each analyte concentration analyzed, for phospho-AKT andphospho-ERK, respectively. Both assays demonstrated sensitivity to lessthan 1 ng/mL.

FIG. 16 shows detection of various concentrations of IL-2 using apeptide tag/anti-peptide tag antibody capture system.

FIG. 17 shows detection of various concentrations of IL-2 using apeptide tag anti peptide tag antibody capture system.

FIG. 18 shows detection of various concentrations of EGF, IL-2 & TNFαusing a peptide tag anti peptide tag antibody capture system.

FIG. 19 shows the signal obtained for various concentrations of analyteusing a peptide tag anti peptide tag antibody capture system.

FIG. 20 shows a comparison of a biotin-streptavidin capture system to apeptide tag-anti-peptide antibody capture system in various biologicalmilieu.

FIG. 21 shows that a streptavidin biotin capture system utilizing anantibody capture agent and an antibody detectable agent is not affectedby increasing concentrations of irrelevant antibodies. shows the datafrom FIG. 21 has been normalised in terms of signal:noise, where noiseis the signal of the immunocomplex obtained for each condition comparedto the signal obtained in the absence of analyte.

FIG. 22A shows that anti peptide tag antibody-peptide capture systemutilizing an antibody capture agent and an antibody detectable agent isnot affected by increasing concentrations of irrelevant antibodies. FIG.22B shows the data from FIG. 22A has been normalised in terms ofsignal:noise, where noise is the signal of the immunocomplex obtainedfor each condition compared to the signal obtained in the absence ofanalyte.

Example 1 Materials

Antibodies used in the following examples include: anti-pERK mousemonoclonal (+/−biotinylation); anti-total ERK rabbit monoclonal(+/−HRP); donkey anti-rabbit-HRP conjugate; anti-S6 p240/44 rabbitpolyclonal (HRP conjugated); anti-S6 mouse monoclonal (biotinylated);anti-AKT pT308 rabbit monoclonal (HRP conjugated); anti-AKT mousemonoclonal (biotinylated); anti-AKT pS473 mouse monoclonal(biotinylated); and anti-AKT rabbit monoclonal (HRP conjugated).

Other reagents and materials used in the following examples include:QuantaRed™ enhanced chemifluorescent HRP substrate (Thermo Scientific);SIGMAFAST™ OPD tablets (Sigma); 96 well clear immunoassay Maxisorp™plates (Nunc); 384 well clear immunoassay Maxisorp™ plates (Nunc);Streptavidin (Sigma); Blocking solution (1% BSA in PBS containing 0.05%polyethylene glycol sorbitan monolaurate sold under the trademark TWEEN20); and A431 cell lysate containing pERK.

Example 2 Methods

1-Wash ELISA Protocol

Nunc 96 well Maxisorp™ plates were passively coated with streptavidinand blocked. pERK cell lysates (50 μL) were added to wells followed bythe addition of a reaction buffer (50 μL) containing pre-optimisedconcentrations of biotinylated anti-pERK mouse mAb and anti-totalERK-HRP rabbit mAb (alternatively a reaction buffer containingbiotinylated anti-pERK mouse mAb, anti-total ERK rabbit mAb andanti-rabbit IgG-HRP can be used).

In certain cases a pre-incubation of pERK cell lysate with theantibodies was performed in a sample plate prior to transfer to thestreptavidin coated plate. Plates were incubated for a minimum of 30 minbefore washing 3× with PBS-T, addition of HRP substrate (100 μL) andmeasurement of product. A similar 1-wash protocol was followed whenusing Nunc 384 well Maxisorp™ plates. The specific kinase antibodieswere supplemented into the protocol and the final reaction volume was 20μL.

Comparative Multi-Wash ELISA Protocol—Streptavidin Coated Plate

Nunc 96 well Maxisorp™ plates were passively coated with streptavidinand blocked. Biotinylated anti-pERK mouse mAb was added to wells andincubated for a minimum of 30 min (100 μL). Plates were washed 3× withPBS-T. pERK cell lysates were added to wells and incubated for a minimumof 30 min (100 μL). Plates were washed 3× with PBS-T. Anti-total ERK-HRPrabbit mAb was added to wells and incubated for a minimum of 30 min (100μL). Plates were washed 3× with PBS-T before addition of HRP substrate(100 μL) and measurement of product.

Comparative Multi-Wash ELISA Protocol—Anti-pERK IgG Coated Plate

Nunc 96 well Maxisorp™ plates were passively coated with anti-pERK mousemAb and blocked. pERK cell lysates were added to wells and incubated fora minimum of 30 min (100 μL). Plates were washed 3× with PBS-T.Anti-total ERK-HRP rabbit mAb was added to wells and incubated for aminimum of 30 min (100 μL). Plates were washed 3× with PBS-T beforeaddition of HRP substrate (100 μL) and measurement of product.

Example 3 Results Assay Characteristics

Speed/Simplicity

In their optimized formats, the 1-wash assay performed comparably to themulti-wash assay in terms of sensitivity (FIG. 1). This was evident onboth streptavidin (Protocols 1 & 2) and anti-pERK IgG (Protocol 3)coated plates for the 30 min (FIG. 1A) and 60 min incubation periods(FIG. 1B). Generally, there was approximately a 10% greater signalobtained at each respective pERK concentration in the 1-wash ELISAcompared to the multi-wash ELISAs but this did not translate to asignificant improvement in the assay detection limit. Importantly, thisdemonstrated that the 1-wash assay could be performed with less handlingsteps and in less than half the time of the multi-wash ELISAs withoutnegatively impacting on sensitivity. This translated to a much simplerELISA assay format by the consolidation of multiple steps into a single1-wash/step system.

Sensitivity

When the 1-wash and multi-wash ELISAs were performed for the same totallength of time of 1 h or less, the 1-wash ELISA was superior insensitivity (FIG. 2). Comparison of a 1×30 min incubation step to 3×10min incubation steps on a streptavidin coated plate (2A) showed that the1-wash system was approximately 10 times more sensitive than themulti-wash system. Although not as significant, this trend was alsonoticeable when comparing a 1×60 min 1-wash assay system on astreptavidin plate, to a 2×30 min multiwash system on an anti-pERK IgGcoated plate (2B). The major benefit of the 1-wash ELISA protocol wasthat it allowed multiple antibody-antigen binding events to occursimultaneously in the single 30 or 60 minute incubation period therebyimproving the pERK detection capabilities per unit time.

Capture Antibody Efficiency

The concentration dependency of anti-pERK IgG (+/−biotinylation) fordetecting pERK was assessed in each of the ELISA protocols (FIG. 3).With or without a pre-incubation step, the 1-wash protocol requiredapproximately 4× and 10× less anti-pERK IgG, to detect the same amountof pERK when compared to multi-wash ELISA protocols 3 and 4respectively. The importance of a pre-incubation step (protocol 1 vsprotocol 2) in the 1-wash ELISA was noticeable when the anti-pERK IgGconcentrations were 100 ng/mL or less. At these lower concentrations,more pERK per unit antibody (approx 15% higher signal) was able to bedetected when a pre-incubation step was incorporated into the 1-washprotocol. Collectively, these results indicated that the 1-wash protocolwas more efficient with its use of anti-pERK IgG compared to themulti-wash format for detecting the same amount of pERK. A possibleexplanation for this phenomenon was that the 1-wash format allowed theformation of solution-phase pERK immune complexes, enabling theirbinding to the streptavidin or anti-pERK IgG coated surface in a moreorientated fashion thereby enhancing antibody functionality. Conversely,in the absence of pERK and detection IgG, biotinylated or unbiotinylatedanti-pERK IgG could bind randomly to the surface, which may have led toa portion of pERK IgG binding sites becoming inaccessible to pERK and/orsterically hindering subsequent binding events in the sandwich (i.e.detection IgG).

The improved anti-pERK IgG efficiency phenomenon highlighted in FIG. 3for the 1-wash ELISA format was investigated further by separating themultiple antibody-antigen binding events of the pERK assay (FIG. 4).This highlighted that independent formation of pERK with anti-totalERK-HRP IgG or anti-pERK IgG (protocols 2 & 3 respectively), prior tobinding to their immobilized partner on the plate, contributed to themore efficient use of anti-pERK IgG in the 1-wash ELISA format.Individually, protocols 2 & 3 were approximately 2 times more efficientwith their use of anti-pERK IgG for detecting pERK compared to themulti-wash ELISA (protocol 4). Furthermore when the individual bindingevents of protocols 2 & 3 were allowed to occur simultaneously as partof the 1-wash ELISA (protocol 1), the use of anti-pERK IgG compared tothe multi-wash procedure was 4-5 times less when measuring the sameconcentration of pERK. Ultimately this highlighted that the binding ofboth antibodies to pERK in solution were important for enhancing thefunctionality of the anti-pERK IgG used in the 1-wash ELISA. This wouldresult in less reagent use (i.e. antibody) and therefore reduced assaycost, compared to the multi-wash ELISA format.

Versatility

The 1-wash ELISA protocol was also challenged using a secondarydetection antibody that was conjugated to HRP (FIG. 5). This wasachieved by replacing the anti-total ERK-HRP with the originalunconjugated antibody (i.e. minus HRP) and introducing anti-rabbitIgG-HRP as the secondary detection antibody. That is, this experimentused a 3 antibody protocol in the 1-wash ELISA format and yielded anA450 signal for pERK of approximately 1.0 AU and a signal:noise value of10. Although unoptimized, in principle this secondary detection approachwas validated in a 1-wash protocol and highlighted the versatility ofthe 1-wash ELISA using at least 3 antibodies.

Robustness

Detection of other phosphoproteins including S6 p240/44, AKT pT308 andAKT pS473 was also achieved in the 1-wash ELISA system (FIG. 6). In 384well streptavidin coated plates, signal:noise ratios of greater than 60were achieved when assaying cell lysates containing the specificphosphoproteins of interest. Like the pERK protocol, the AKT pS473 assayalso used an anti-phospho IgG as the capture antibody with an anti-totalIgG used as the detection antibody (i.e. conjugated to HRP).Alternatively the S6 p240/44 and AKT pS473 assays used an anti-total IgGas the capture antibody, with a specific anti-phospho IgG-HRP completingthe sandwich. These results demonstrated the robustness of the 1-washELISA with its ability to detect different targets in varying immunecomplex orientations.

Example 4 Microfluidics

Microfluidic reactions were performed in a microfluidic cartridge asshown in FIG. 7.

Referring to FIG. 7, the microfluidic cartridge 700 comprises a plasticsubstrate 710 into which a plurality of flow channels 730 are formed. Asample is introduced into the flow channel 730 via sample inlet 720. Thesample is then driven along flow channel 730 by a pump (not shown).Detection region 740 comprises an electrode for electrochemicaldetection to which an immobilization agent is bound. In the embodimentsdescribed in the following examples, the immobilization agent isstreptavidin. Moreover, although the present invention contemplates anysuitable electrodes and methods for electrochemical detection, themethod described in the following examples utilizes the electrodes anddetection methods described in U.S. Pat. No. 6,770,190. After passingover detection area 740, the sample is transported to waste collectionarea 750.

An example of the method of the present invention performed in themicrofluidic cartridge is described below:

Samples were mixed with a reaction buffer (phosphate buffered saline,BSA 0.3%, polyethylene glycol sorbitan monolaurate sold under thetrademark TWEEN 20 0.1%) containing two antibodies to the analyte ofinterest. For each analyte, the two antibodies were raised againstdistinct epitopes on the analyte of interest, such that both antibodiescould bind to the protein of interest simultaneously. One of theantibodies performed the function of a capture agent and had biotinattached to it, while the other antibody performed the function of adetectable agent and was linked to horse radish peroxidise (HRP).

The samples being measured contained varying amounts of an analyte ofinterest, in the present examples either phospho-ERK or phospho-AKT. Amicrofluidic cartridge (see FIG. 7) was placed on a pumping anddetection instrument, and samples were drawn onto the microfluidiccartridge into separate lanes of the cartridge. The cartridge bound thebiotinylated antibody at the detection region. As set out above, thedetection region comprised an electrode for electrochemical detection towhich streptavidin is bound as an immobilisation agent. As such, acomplex comprising biotinylated capture antibody, bound analyte andHRP-linked detectable antibody would become immobilised to the electrodevia interaction of the biotin on the capture antibody and streptavidinon the electrode.

After capture, the cartridge was automatically washed with bufferwithout antibodies. Following this wash step, a solution containing HRPsubstrate (SigmaFAST OPD) was drawn over the cartridge, allowing boundHRP to convert the HRP substrate to products that could be detectedelectrochemically by the electrode and detection equipment present onthe pumping device. The electrical signals generated were proportionalto the level of HRP-induced product conversion, which was proportionalto the amount of analyte bound to the capture antibodies.

Example 5 Detection of pERK Using a Microfluidic System

Recombinant pERK was diluted in 1× lysis buffer, with four folddilutions from a top concentration of 400 ng/ml (10 nM). Samples werepre incubated with an equal volume of reaction buffer (see above).

The sample/reaction buffer mix was then run on a microfluidic cartridgeas described in Example 4. The results are shown in FIGS. 8A-8C. Eachdata point shown is the average of 3 flow cells from a single cartridge.The data was transformed by taking the point at which substrateinjection begins as zero. Data was collected from the point at whichsubstrate flow through begins up until the end of substrate incubationphase (180 s after substrate injection).

As can be seen by comparing FIGS. 8 b and 8 c, data collection at theend of the substrate incubation phase (180 s after substrate injection)appeared to provide greater sensitivity. Using the data taken from 180seconds after injection of substrate, the detection limit of the chipwas about 2 ng/ml pERK.

Example 6 Detection of pAKT Using a Microfluidic System

Recombinant pAKT473 was diluted in 1× lysis buffer, with five folddilutions from a top concentration of 100 ng/ml. Samples were preincubated for two hours with an equal volume of reaction buffer (seeabove) to equilibrate the interaction and so minimise incubation effectsduring the run.

The sample/reaction buffer mix was then run on a microfluidic cartridgeas described in Example 4. The results are shown in FIGS. 9A-9C. Eachdata point shown is the average of 3 flow cells from a single cartridge.The data was transformed by taking the point at which substrateinjection begins as zero. Data was collected from the point at whichsubstrate flow through begins up until the end of substrate incubationphase (180 s after substrate injection).

Using the data taken from 180 seconds after injection of substrate, thedetection limit of the chip was about 1 ng/ml pAKT.

Example 7 Reagent Order of Addition Permutations

Capture antibody (anti-pERK-peptide conjugate or anti-pERK-biotinconjugate), detection antibody (anti-total ERK-HRP conjugate),capture/detection antibody mixture, and varying concentrations of celllysate containing pERK were added to (A) anti-peptide conjugate antibodycoated plates or (B) streptavidin coated microplates, in 8 differentpermutations (refer to Table 1 & 2). Individual assay components wereadded 1 min apart to the plates, and incubated for 2 h. Plates werewashed, incubated with HRP substrate, before detection of thefluorescent product.

TABLE 1 Reagent volumes for order of addition assessment Assay ComponentVolume/Well Capture/Detection Antibody Mix 50 μl Lysate 50 μl CaptureAntibody 25 μl Detection Antibody 25 μl

TABLE 2 Reagent order of addition permutations Trial # 1^(st) Addition2^(nd) Addition 3^(rd) Addition 1 Capture/Detection Ab Mix Lysate n/a 2Lysate Capture/Detection Ab n/a Mix 3 Lysate Capture Ab Detection Ab 4Lysate Detection Ab Capture Ab 5 Detection Ab Capture Ab Lysate 6Detection Ab Lysate Capture Ab 7 Capture Ab Lysate Detection Ab 8Capture Ab Detection Ab Lysate

The effect of reagent order of addition on pERK detection in thesingle-incubation ELISA using different capture systems is shown in FIG.10. Across the 8 different permutations, and at several analyteconcentrations, little signal difference were observed. This resultdemonstrates that equivalent results can be obtained in asingle-incubation ELISA assay, irrespective of the order of addition ofthe individual components.

Example 8 Recombinant Protein Standard Curves in Different BiologicalMilieu Using the Peptide Conjugate Capture System

A demonstration of the use of the single-incubation ELISA assay formatfor the detection of three recombinant human proteins diluted in humanserum is provided in FIG. 11. EGF, IL-2 and TNFα were measured inPBS/0.5% BSA and human serum. Detection limits of ≦10 pg/mL wereascertained for each assay in PBS/0.5% BSA, and similar sensitivity forboth IL-2 and TNFα were observed for analyte diluted in human serum. Thedetection limit for EGF in human serum could not be detected due to thepresence of a high level of endogenous EGF, which was confirmed using astandard commercial EGF ELISA kit (R&D Systems, data not shown). FIG. 11shows the mean and standard deviations for the duplicate data points foreach target analyzed.

This data illustrates that the single-incubation ELISA assay format wasrobust to measuring analytes in different biological milieu. The assayclearly demonstrates efficacy for several different targets in serum,whereby the assay components are incubated concurrently. The high signalfor EGF in human serum is due to the presence of endogenous EGFprotein(s) in this medium.

Example 9 Recombinant Protein Standard Curves Using the PeptideConjugate Capture System in a 10 Min Single-Incubation ELISA

Nunc 96 well Maxisorp™ plates were passively coated with an anti peptidetag antibody overnight at 4° C. Plates were washed 3× with PBS-T andblocked with 200 μL/well of a 1% BSA solution in PBS-T (0.05%). Blockingsolution was aspirated prior to assay. Analyte (eg 50 μL of recombinantprotein) were added to the wells followed by the addition of an antibodyantibody mixture (50 μL) containing pre-optimised concentrations ofpeptide tag conjugated anti-analyte capture antibody and HRP-conjugatedanti-analyte detection antibody. Plates were incubated for 10 min beforewashing 3× with PBS-T. Fluorescent HRP substrate (100 μL) was added tothe wells and incubated for 5 mins before measurement of fluorescentproduct.

Recombinant human proteins EGF, IL-2 and TNFα were prepared in PBS/0.5%BSA at concentrations ranging from 100 ng/mL down to 1 pg/mL and 50μL/well added to an anti peptide tag antibody ELISA plate.Capture/detection antibody mix for EGF (A), IL-2 (B) and TNFα (C) wereadded to the appropriate ELISA plate wells and incubated for 10 min.Plates were washed before incubation with HRP substrate for 5 min anddetection of the fluorescent product.

FIG. 12 shows the detection of three recombinant human proteins using a10 min single-incubation ELISA assay format on an anti peptide tagantibody coated ELISA plate. EGF, IL-2 and TNFα standard curves weremeasured successfully in PBS/0.5% BSA with detection limits of ≦32 pg/mLascertained for each assay. This data illustrated that the simplifiedpeptide conjugate capture/single-incubation ELISA assay format wasamenable to measuring multiple analytes on the same plate in as littleas 10 minutes. As can be seen, in the 10 minute single-incubation ELISAthe assay was still able to efficiently detect the three analytes, evenat a concentration of the analytes less than 100 pg/ml.

Example 10 Intra-Plate Variation

FIG. 13 shows intra-plate variation observed for 2 separatesingle-incubation ELISAs for either phospho-AKT (pSer473) orphospho-STAT3. For each target, cellular lysate was diluted to 3different concentrations using 1× Lysis buffer as indicated, and addedto 24 replicate wells of a 96-well streptavidin-coated microplate. Toinitiate the assay reaction, for either target, a mixture of thebiotin-conjugated capture antibody, and the HRP-conjugated detectionantibody were added to the lysates, and incubated for 1 hour. The wellswere subjected to a standard wash cycle for each assay. After the washcycle, QuantaRed™ HRP substrate was added to the wells, and each platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 550ex/600em nm. FIGS. 13A and 13B show the datapoints at each lysate concentration analyzed, for phospho-AKT andphospho-STAT3, respectively. The coefficient of variation (CV %) foreach analyte concentration was calculated by dividing the standarddeviation observed over the 24 wells at each concentration, by the meanof the 24 wells at the same concentration, and transforming thisfraction to a percentage value. Typically, a value of less than 10% isdesired for many assays, for example, in certain high quality assays,and the data presented here demonstrates suitable low intra-platevariability characteristics.

Example 11 Detection of TNFα

FIG. 14 shows detection of TNFα in tissue culture supernates. THP-1cells were seeded into 96-well tissue culture microplates in RMPI cellculture medium containing 10% (v/v) foetal bovine serum and variousother standard cell culture additives. The cells were then treated witha various concentrations of PMA diluted in the same medium, andincubated overnight in a humidified 37° C. incubator. The following day50 μL of medium was aspirated from the cell culture wells, and added tothe wells of a peptide-coated 96-well assay plate. The assay reactionwas initiated by the addition of 50 μL of an antibody mixture containingthe capture antibody-peptide conjugate, and the detection antibody-HRPconjugate, and incubated for 1 hour. The wells were subjected to astandard wash cycle for each assay. After the wash cycle, fluorescentHRP substrate was added to the wells, and each plate was incubated for10 min in the dark. The fluorescent signal in the wells was measured at540ex/590em nm, and quantitated using a standard curve generated againstthe same target. FIG. 14 shows the mean and standard deviations for theduplicate data points for each target analyzed. In this Figure, theassay demonstrates efficient detection of specific target analyte intissue culture supernates using the certain embodiments, whereby theassay components are incubated concurrently.

Example 12 Detection of Phospho-AKT (pSer473) or Phospho-ERK in a 25 MinTotal Assay Time

FIG. 15 shows detection of either phospho-AKT (pSer473) or phospho-ERKin a 25 min total assay time. For each target, recombinant active (A)phospho-AKT or (B) phospho-ERK was diluted as indicated, to variousconcentrations using 1× Lysis buffer containing 0.1% BSA and added to 4replicate wells of a 96-well streptavidin-coated microplate. To initiatethe assay reaction, for either target, a mixture of thebiotin-conjugated capture antibody, and the HRP-conjugated detectionantibody were added to the lysates, and incubated for 1 hour. The wellswere subjected to a standard wash cycle for each assay. After the washcycle, QuantaRed™ HRP substrate was added to the wells, and each platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 550ex/600em nm. FIGS. 15A and 15B show the datapoints at each analyte concentration analyzed, for phospho-AKT andphospho-ERK, respectively. Both assays demonstrated sensitivity to lessthan 1 ng/mL.

Example 13 Detection of IL-2 in Using a ERK Peptide-Anti Peptide CapturePair

FIG. 16 shows detection of IL-2 in using a ERK peptide-anti peptidecapture pair. Recombinant interleukin 2 (IL-2) was diluted as indicated,to various concentrations using 1×PBS containing 0.1% BSA and added toduplicate wells of a 96-well anti-ERK-peptide antibody-coatedmicroplate. To initiate the assay reaction, for either target, a mixtureof the ERK peptide capture antibody, and the HRP-conjugated detectionantibody were added to the lysates, and incubated for 1 hour. The wellswere subjected to a standard wash cycle for each assay. After the washcycle, fluorescent HRP substrate was added to the wells, and each platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 540ex/590em nm. FIG. 16 shows the data points ateach analyte concentration analyzed, demonstrating sensitivity to 100pg/mL or less.

Example 14 General Discussion

The single-incubation ELISA uses an immuno-sandwich format, but with atleast one difference. For the single-incubation ELISA assay, both theanalyte and the assay reagents are added to the assay microplate at thesame time, in solution. After a short incubation period, unbound assayreagents and analytes are washed away, and immuno-complexes containingboth antibodies are detected. The single-incubation ELISA allows theuser a higher degree of assay flexibility. In contrast to other ELISAformats, in particular sets of examples no target-specific antibodiesare present on the assay microplate itself, so assays for severaldifferent targets can be performed in different wells on the samemicroplate. For example, a cellular lysate can be analyzed on the sameassay microplate in parallel for p38-MAPK phosphorylation, ERKphosphorylation, AKT phosphorylation and JNK phosphorylation, givingfast, accurate and quantifiable information on key cell signallingevents. However, if desired target antibodies may be immobilized on theplate.

The single-incubation ELISA provides the high quality results desiredfrom a sandwich immunoassay, and the assay allows for the use ofself-contained kits to conduct the assay.

For example, a kit may contain one or more of the following components:

Capture Antibody Reagent

Detection Antibody Reagent

Lysis Buffer (for example supplied at 5× concentration) containing amixture of detergents for cellular lysis, and phosphatase inhibitors.

Enhancer Solution—containing factors for enhancing assay performance,such as anti-HAMA components, and target-specific additives to increaseassay performance.

ADHP Dilution Buffer—containing cofactors necessary for the HRP-mediatedconversion of ADHP to resorufin.

ADHP (for example supplied at 100× concentration)

Wash Buffer (for example supplied at 10× concentration)

Stop Solution—for stopping HRP activity when necessary

Assay Control Lysate

Assay microplate

Assay diluent—for the dilution of concentrated samples

Example 15 General Assay Protocols

(i) Protocol for use with samples such as cellular lysates and tissueculture supernates

Assay Protocol

1. Add 50 μl/well of sample to the assay microplate. 50 μl/well assaycontrols may be added to separate wells if desired.

2. Add 50 μl/well of antibody mix to the wells. Generally aconcentration of antibodies in the mix of 50-500 ng/mL is suitable.Cover the microplate and incubate at room temp on a microplate shaker(−300 rpm).

3. Wash wells with 200 μl/well wash buffer (repeat 3 times). After finalwash, remove any remaining wash solution from wells. A suitable washbuffer is PBS containing polyethylene glycol sorbitan monolaurate soldunder the trademark TWEEN 20.

4. Immediately prior to use, prepare substrate mix. A suitable substratemix is TMB, ADHP, OPD, or other suitable HRP substrates, diluted withco-factors suitable for mediating their conversion to measurableby-products. Add 100 μl/well of substrate mix. Cover microplate withfoil, and incubate for 10 minutes at room temp on a microplate shaker(−300 rpm).

5. Add 10 μl/well stop solution, and mix briefly (5-10 sec) on amicroplate shaker. A suitable stop solution is a dilute acid such asHCl, or a strong detergent such as SDS.

6. Read fluorescence signal with a compatible filter set.

(ii) Protocol for serum samples, or other samples that may carrysample-specific interferences

Assay Protocol

1. Add 25 μl/well Enhancer mix. Enhancer mix containing generalcomponents for the neutralization of HAMAs, as well other components forthe neutralization of target-specific binding proteins carried in serum.

2. Add 50 μl/well of sample to the assay microplate. 50 μl/well assaycontrols may be added to separate wells if desired.

3. Add 25 μl/well of antibody mix to the wells. Cover the micro plateand incubate for 1 hour at room temp on a microplate shaker (−300 rpm).

3. Wash wells with 200 μl/well wash buffer (repeat 3 times). After finalwash, remove any remaining wash solution from wells.

4 Prepare substrate prior to use and add 100 μl/well. Cover microplatewith foil, and incubate for 10 minutes at room temp on a microplateshaker (−300 rpm).

5. Add 10 μl/well stop solution, and mix briefly (5-10 sec) on amicroplate shaker.

6. Read fluorescence signal with a compatible filter set.

Example 16 Detection of IL-2 in Using a ERK Peptide-Anti Peptide CapturePair

FIG. 16 shows detection of IL-2 in using a ERK peptide-anti peptidecapture pair. Recombinant interleukin 2 (IL-2) was diluted as indicated,to various concentrations using 1×PBS containing 0.1% BSA and added toduplicate wells of a 96-well anti-ERK-peptide antibody-coatedmicroplate. To initiate the assay reaction, for either target, a mixtureof the ERK peptide capture antibody, and the HRP-conjugated detectionantibody were added to the lysates, and incubated for 1 hour. The wellswere subjected to a standard wash cycle for each assay. After the washcycle, fluorescent HRP substrate was added to the wells, and each platewas incubated for 10 min in the dark. The fluorescent signal in thewells was measured at 540ex/590em nm. FIG. 16 shows the data points ateach analyte concentration analyzed, demonstrating sensitivity to 100pg/mL or less.

Example 17 Detection of Various Concentrations of IL-2 Using a PeptideTag Anti Peptide Tag Antibody Capture System

FIG. 17 shows detection of various concentrations of IL-2 using apeptide tag anti peptide tag antibody capture system.

Antibodies were generated in mice as monoclonal antibodies to a 23 aminoacid peptide, KRITVEEALAHPYLEQYYDPTDE (SEQ ID NO. 2), a sequence derivedfrom the carboxy terminus of the human ERK proteins (ERK C-termpeptide). Purified antibodies (TGR, 12D4) to this peptide were passivelycoated onto a maxisorb Nunc immunoassay plate, and the plate thenblocked against further non-specific protein attachment. The ERK C-termpeptide was also used to conjugate to antibodies to the human IL-2protein (R&D Systems), so that the peptide would act to anchor thisantibody to the plate surface. A second IL-2 antibody (R&D Systems) wasconjugated to horse radish peroxidase (HRP) to be used as the reporterantibody. Recombinant human IL-2 was mixed with PBS/BSA (0.1%) atvarious concentrations shown, and to these solutions were added the IL-2antibodies. After an hour incubation, the wells were washed with a washbuffer, and fluorescent HRP substrate added for 10 min, followed byreading of the plate at 540/590 nm ex/em wavelengths in a plate reader.

It can be seen that the assay system measured the concentrations of IL-2present in each sample and that the variation between samples was low asindicated by the small error bars.

Example 18 Detection of Various Concentrations of EGF, IL-2 & TNFα Usinga Peptide Tag Anti Peptide Tag Antibody Capture System

FIG. 18 shows detection of various concentrations of EGF, IL-2 & TNFαusing a peptide tag anti peptide tag antibody capture system.

Antibodies specific to the peptide DYKDDDDK (SEQ ID NO. 1; Sigma,catalog number F1804) were passively coated onto a maxisorb Nuncimmunoassay plate at 5 μg/mL overnight in PBS, and the plate thenblocked against further non-specific protein attachment. The peptideDYKDDDDK (SEQ ID NO. 1) was also used to conjugate to IgG antibodies tothe human IL-2 protein (R&D Systems), human EGF or human TNF□ so thatthe peptide would act to anchor this antibody to the plate surface. Asecond detectable antibody to each analyte (R&D Systems) was alsoconjugated to horse radish peroxidase (HRP) to be used as the reporterantibody. EGF, IL-2 & TNFα peptide (C-terminal acid) capture IgG's & andtheir respective HRP detection were IgG's prepared in reaction buffer.Pure analytes as standards were diluted in PBS/BSA (0.5%) at variousconcentrations shown. Analyte (50 μL/well) was added to the coated plateand then added 50 μL/well of corresponding antibody mix (Capture 200ng/mL; detection 50 ng/mL). After an hour incubation with shaking, thewells were washed three time with a wash buffer, and fluorescent HRPsubstrate (ADHP) added for 10 min, followed by reading of the plate at540/590 nm ex/em wavelengths in a plate reader. The data shows thesensitive detection of each of EGF, IL-2 and TNF□ in separate wells of amicrotitre plate using a single-wash, peptide tag antibody capturesystem.

Example 19 Detection of Various Concentrations of Analyte Using aPeptide Tag Anti Peptide Tag Antibody Capture System

FIG. 19 shows the signal obtained for various concentrations of analyteusing a peptide tag anti peptide tag antibody capture system.

Antibodies were generated in mice as monoclonal antibodies to thepeptide DYKDDDDK (SEQ ID NO. 1). Purified antibodies to this peptidewere coated onto a maxisorb Nunc immunoassay plate at 10 ug/ml, and theplate then blocked against further non-specific protein attachment. Thepeptide DYKDDDDK (SEQ ID NO. 1) was also used to conjugate to antibodiesto the human TNFα protein (R&D Systems), so that the peptide would actto anchor this antibody to the plate surface. A second TNF□ antibody(R&D Systems) was conjugated to horse radish peroxidase (HRP) to be usedas the reporter antibody. TNFα was mixed with PBS/BSA (0.5%) at variousconcentrations shown, and to these solutions were added the IL-2antibodies (Capture 200 ng/mL; detection 50 ng/mL). After an hourincubation with shaking, the wells were washed with a wash buffer, andfluorescent HRP substrate added for 10 min, followed by reading of theplate at 540/590 nm ex/em wavelengths in a plate reader. The data showsthat the use of a peptide tag antibody capture system, whereby in thiscase the peptide tag was DYKDDDDK, and the system was a single-washELISA format, enabled the sensitive measurement of TNFα with a totalassay time of approximately 1 hour.

Example 20 Comparison of a Biotin-Streptavidin Capture System to aPeptide Tag-Anti-Peptide Antibody Capture System in Various BiologicalMilieu

FIG. 20 shows a comparison of a biotin-streptavidin capture system to apeptide tag-anti-peptide antibody capture system in various biologicalmilieu.

Antibodies were generated in mice as monoclonal antibodies to thepeptide DYKDDDDK (SEQ ID. NO. 1). Purified antibodies to this peptidewere coated onto a maxisorb Nunc immunoassay plate at 10 μg/ml overnightin carbonate buffer, and the plate washed and then blocked againstfurther non-specific protein attachment. Separately, a commercialstreptavidin-coated plate (Nunc Immobiliser) was used forbiotin-conjugated antibodies assays. The peptide DYKDDDDK (SEQ ID NO. 1)was used to conjugate to antibodies to the human TNFα protein (R&DSystems), so that the peptide would act to anchor this antibody to theplate surface to which had been coated antibodies to this peptide.Separately, antibodies to the human TNFα protein (R&D Systems), werealso linked with biotin, so that this would act to anchor this antibodyto the plate surface to which had been coated streptavidin. A secondspecies of TNFα antibody (R&D Systems) was conjugated to horse radishperoxidase (HRP) to be used as the reporter antibody. TNFα was mixedwith various media (blocking buffer, milk, human serum, FBS, urine orRPMI) at 100 pg/mL or not added at all, and to these solutions wereadded either to the TNFα antibodies linked with biotin (Capture 750ng/mL; detection 50 ng/mL) or peptide DYKDDDDK (SEQ ID NO. 1) (Capture300 ng/mL; detection 50 ng/mL), and the HRP-linked TNFα antibodies.After an hour incubation, the wells were washed with a wash buffer, andfluorescent HRP substrate ADHP added for 10 min, followed by reading ofthe plate at 540/590 nm ex/em wavelengths in a plate reader. It can beseen from the data that the peptide tag-anti-peptide antibody capturesystems was superior to the biotin-streptavidin system in detectinganalytes, particularly when analytes were present in particular media.Of special note are the inhibitory effects on the assay of TNFα presentin milk, serum, FBS and RPMI when using the biotin-streptavidin system,reflecting the presence of biotin in these samples that interferes withthis capture system.

Example 21 Streptavidin Biotin Capture Systems Utilizing an AntibodyCapture Agent and an Antibody Detectable Agent is not Affected byIncreasing Concentrations of Irrelevant Antibodies

FIG. 21 shows that a streptavidin biotin capture system utilizing anantibody capture agent and an antibody detectable agent is not affectedby increasing concentrations of irrelevant antibodies.

Nunc Immobiliser plates, coated with streptavidin, were used in an assayto determine capacity of p-ERK antibody binding and p-ERK analytemeasurement. Antibodies to the phosphorylation site of the ERK protein(TGR, Thr202/Tyr204) were linked with biotin, so that this would act toanchor this antibody to the plate surface to which has been coatedstreptavidin. Separately, a second ERK antibody (Santa Cruz) was linkedto horse radish peroxidase (HRP) to act as a reporter antibody. Samplescontaining cellular lysates in which the p-ERK protein was present atvarious concentrations were then mixed with the ERK antibodies either inthe absence (1-plex) or presence (4-12-plex) of increasing numbers ofpairs of irrelevant antibodies at the same concentration as the ERKantibodies, such that one of the pair of the irrelevant antibodies wasalso biotinylated in the same way and extent as the ERK antibody. After1 hour, the wells were washed with a wash buffer, and fluorescent HRPsubstrate ADHP added for 10 min, followed by reading of the plate at540/590 nm ex/em wavelengths in a plate reader. Results are presented asabsolute fluorescence signal.

FIG. 21 shows the data normalised in terms of signal:noise, where noiseis the signal of the immunocomplex obtained for each condition comparedto the signal obtained in the absence of analyte.

It can be seen from these graphs that the single-wash assay system withboth antibodies being present with the analyte, in this case using thebiotin-streptavidin pair, can use low concentrations of Captureantibodies, allowing the presence of up to 12 pairs of unrelated taggedantibodies to be present without there being any assay interference.

Example 22 Anti Peptide Tag Antibody-Peptide Capture Systems Utilizingan Antibody Capture Agent and an Antibody Detectable Agent is notAffected by Increasing Concentrations of Irrelevant Antibodies

FIG. 22A shows that anti peptide tag antibody-peptide capture systemutilizing an antibody capture agent and an antibody detectable agent isnot affected by increasing concentrations of irrelevant antibodies

Antibodies were generated in mice as monoclonal antibodies to thepeptide DYKDDDDK (SEQ ID NO. 1). Purified antibodies to this peptidewere coated onto a maxisorb Nunc immunoassay plate, and the plate thenblocked against further non-specific protein attachment. Antibodies tothe human EGF protein (R&D Systems) were linked with the peptideDYKDDDDK (SEQ ID NO. 1), so that this would act to anchor this antibodyto the plate surface to which has been coated streptavidin. Separately,a second EGF antibody (R&D Systems) was linked to horse radishperoxidase (HRP) to act as a reporter antibody. Samples containing EGFat various concentrations were then mixed with the EGF antibodies eitherin the absence (1-plex) or presence (4-12-plex) of increasing numbers ofpairs of irrelevant antibodies at the same concentration as the EGFantibodies, such that one of the pair of the irrelevant antibodies wasalso linked with the peptide DYKDDDDK (SEQ ID NO. 1) in the same way andextent as the EGF antibody. After 1 hour, the wells were washed with awash buffer, and fluorescent HRP substrate ADHP added for 10 min,followed by reading of the plate at 540/590 nm ex/em wavelengths in aplate reader. Results are presented as absolute fluorescence signal.

FIG. 22B shows the data from FIG. 22A normalised in terms ofsignal:noise, where noise is the signal of the immunocomplex obtainedfor each condition compared to the signal obtained in the absence ofanalyte.

It can be seen from these graphs that the single-wash assay system withboth antibodies being present with the analyte, in this case using thepeptide-anti-peptide antibody pair, can use low concentrations ofCapture antibodies, allowing the presence of up to 12 pairs of unrelatedtagged antibodies to be present without there being any assayinterference.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto, or indicated in this specification, individually or collectively,and any and all combinations of any two or more of the steps orfeatures.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Also, it must be noted that, as used herein, the singular forms “a”,“an” and “the” include plural aspects unless the context alreadydictates otherwise.

The invention claimed is:
 1. A method for detecting an analyte,comprising: covalently binding an antibody capture agent with aplurality of peptide tags to form a tagged antibody capture agent;forming an analyte complex comprising the analyte bound to the taggedantibody capture agent and to a detectable agent separately; introducingthe analyte complex to an anti-peptide antibody immobilized on asubstrate; immobilizing the analyte complex to the substrate by bindingat least one of the plurality of peptide tags with the anti-peptideantibody; and detecting the analyte by detection of the detectableagent.
 2. The method according to claim 1, wherein the analyte is anon-nucleic acid analyte.
 3. The method according to claim 1, whereinthe tagged antibody capture agent complexes with a non-nucleic acidportion of the analyte.
 4. The method according to claim 1, wherein thetagged antibody capture agent binds with the analyte substantiallyexclusive of nucleic acid-nucleic acid interactions.
 5. The methodaccording to claim 1, wherein at least one of the plurality of peptidetags is not naturally occurring.
 6. The method according to claim 1,wherein the covalently binding comprises cross-linking.
 7. The methodaccording to claim 6, wherein the cross-linking comprises covalentconjugation by primary amines in the antibody capture agent.
 8. Themethod according to claim 1, wherein the tagged antibody capture agentis in solution.
 9. The method according to claim 8, wherein the solutionhas a tagged antibody capture agent concentration in the range from 50to 500 ng/ml.
 10. The method according to claim 1, wherein the taggedantibody capture agent has a dissociation constant, Kd, of between 10⁻⁶and 10⁻¹² M.
 11. The method according to claim 1, wherein the detectableagent is in solution.
 12. The method according to claim 1, wherein thedetectable agent comprises a detectable tag.
 13. The method according toclaim 12, wherein the detectable tag comprises one or more of an enzyme,a fluorophore, and a lanthanide ion.
 14. The method according to claim1, wherein the anti-peptide antibody is bound to the substrate via anon-covalent attachment.
 15. The method according to claim 1, whereinthe substrate is at least partially blocked.
 16. The method according toclaim 1, wherein the complexing occurs in a solution.
 17. The methodaccording to claim 16, wherein the solution has a tagged antibodycapture agent concentration in the range from 50 to 500 ng/ml.
 18. Themethod according to claim 1, wherein the analyte is provided in abiological sample.
 19. The method according to claim 18, wherein thesample has an analyte concentration of 100 ng/ml or less.
 20. The methodaccording to claim 18, wherein the sample comprises one or more proteinsseparate from the analyte wherein the one or more proteins have a totalconcentration of more than 0.01 mg/ml.
 21. The method according to claim19, wherein the sample comprises one or more proteins separate from theanalyte wherein the one or more proteins have a total concentration ofmore than 0.01 mg/ml.
 22. The method according to claim 18, wherein thesample comprises one or more of blood, serum, urine, milk, cell lysate,and a derivative thereof.
 23. The method according to claim 21, whereinthe sample comprises one or more of blood, serum, urine, milk, celllysate, and a derivative thereof.
 24. The method according to claim 1,wherein the complexing and the immobilizing occur in a single reactionvessel, and more than one analyte is detected in the single reactionvessel.
 25. The method according to claim 1, wherein the complexingoccurs in a first reaction vessel and the immobilizing occurs in asecond reaction vessel.
 26. The method according to claim 1, wherein theimmobilizing comprises incubating.
 27. The method according to claim 26,wherein the analyte is detected in less than 120 minutes after theincubating is initiated.
 28. The method according to claim 1, furthercomprising one or more washings after immobilizing.
 29. The methodaccording to claim 28, wherein the one or more washings consists of nomore than one washing.
 30. The method according to claim 1, wherein theplurality of peptide tags comprises a peptide comprising the amino acidsequence DYKDDDDK (SEQ ID NO. I).